U.S. patent application number 12/767724 was filed with the patent office on 2011-03-03 for regulation of oncogenes by micrornas.
This patent application is currently assigned to Yale University. Invention is credited to Helge Grosshans, Steven M. Johnson, Frank J. Slack, Joanne Barnes Weidhaas.
Application Number | 20110054006 12/767724 |
Document ID | / |
Family ID | 36036868 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110054006 |
Kind Code |
A1 |
Slack; Frank J. ; et
al. |
March 3, 2011 |
REGULATION OF ONCOGENES BY MICRORNAS
Abstract
Naturally occurring miRNAs that regulate human oncogenes and
methods of use thereof are described. Suitable nucleic acids for
use in the methods and compositions described herein include, but
are not limited to, pri-miRNA, pre-miRNA, mature miRNA or fragments
of variants thereof that retain the biological activity of the
mature miRNA and DNA encoding a pri-miRNA, pre-miRNA, mature miRNA,
fragments or variants thereof, or regulatory elements of the miRNA.
The compositions are administered to a subject prior to
administration of a cytotoxic therapy in an amount effective to
sensitize cells or tissues to be treated to the effects of the
cytotoxic therapy.
Inventors: |
Slack; Frank J.; (Branford,
CT) ; Johnson; Steven M.; (Redwood City, CA) ;
Grosshans; Helge; (Basel, CH) ; Weidhaas; Joanne
Barnes; (Westport, CT) |
Assignee: |
Yale University
New Haven
CT
|
Family ID: |
36036868 |
Appl. No.: |
12/767724 |
Filed: |
April 26, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11876503 |
Oct 22, 2007 |
7741306 |
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12767724 |
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11219379 |
Sep 2, 2005 |
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11876503 |
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60606855 |
Sep 2, 2004 |
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60853061 |
Oct 20, 2006 |
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60931740 |
May 25, 2007 |
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Current U.S.
Class: |
514/44A |
Current CPC
Class: |
A61P 1/16 20180101; A61P
1/18 20180101; C12N 15/1135 20130101; A61K 48/00 20130101; C12N
2310/141 20130101; A61P 13/08 20180101; C12N 15/1138 20130101; A61P
35/00 20180101; A61P 5/00 20180101; A61P 35/02 20180101; A61K 41/00
20130101; A61P 21/00 20180101; A61P 15/00 20180101; C12N 15/1136
20130101; A61P 25/00 20180101; A61P 13/10 20180101; A61K 38/00
20130101; A61P 17/00 20180101; A61P 1/02 20180101; A61P 11/00
20180101; C12N 2310/111 20130101; A61K 31/7088 20130101; A61P 19/00
20180101; A61P 43/00 20180101; A61P 1/04 20180101; A61K 45/06
20130101; A61K 31/7088 20130101; A61K 2300/00 20130101; A61K 41/00
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/44.A |
International
Class: |
A61K 31/7088 20060101
A61K031/7088; A61P 35/00 20060101 A61P035/00 |
Goverment Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
[0002] The Federal Government has certain rights in this invention
by virtue of Grant No. 1R01GM062594-01A1 and Grant No.
1R01GM064701-01 from the National Institutes of Health to Frank J.
Slack.
Claims
1. A method for increasing the sensitivity of a cell to cytotoxic
therapy comprising administering one or more miRNAs to a patient,
and subsequently administering a chemotherapeutic agent or
radiation, wherein the one or more miRNAs are administered in an
amount effective to sensitize the cells or tissues to be treated to
the cytotoxic therapy.
2-24. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
11/876,503, filed Oct. 22, 2007, entitled "Regulation of Oncogenes
by MicroRNAs," now U.S. Pat. No. 7,741,306, which is a continuation
in part of U.S. Ser. No. 11/219,379, filed Sep. 2, 2005, entitled
"Regulation of Oncogenes by MicroRNAs" which claims priority to
U.S. Ser. No. 60/606,855 entitled "Regulation of Oncogenes by
MicroRNAs" filed Sep. 4, 2004, and also claims priority to U.S.
Ser. No. 60/853,061 entitled "MicroRNA Manipulation to Alter the
Radiation Response" filed Oct. 20, 2006 and U.S. Ser. No.
60/931,740 entitled "A Role for MicroRNA Manipulation in Altering
Cellular Radiation Resistance" filed May 25, 2007.
BACKGROUND OF THE INVENTION
[0003] Cancer is a group of diseases characterized by uncontrolled
growth and spread of abnormal cells. Cancer is caused by both
external factors (tobacco, chemicals, radiation, and infections
organisms) and internal factors (inherited mutations, hormones,
immune conditions, and DNA damage). These factors may act together
or sequentially to initiate and/or promote carcinogenesis. Cancer
causes 1 of every 4 deaths and is the leading cause of death in
people under age 85 in the United States. Nearly half of all men
and a little over one third of all women in the U.S. will develop
cancer during their lifetimes. Today, millions of people are living
with cancer or have had cancer. The sooner a cancer is found and
treatment begins, the better are the chances for living for many
years.
[0004] Lung cancer is the leading cause of cancer deaths for both
men and women in the United States. According to the American
Cancer Society, about 160,000 people die annually of this disease,
with about 170,000 newly diagnosed cases each year. Despite the use
of surgery, chemotherapy, and radiation, the survival rate for
patients remains extremely poor (>15% over 5 years). As
estimated by the American Cancer Society, the 5-year survival rate
for all cancers is about 64% for cancers diagnosed between
1995-2000. However, the survival rate varies depending on cancer
type and the stage of cancer at time of detection. For example, the
survival rate for brain, breast, and colon cancer is 33, 88, and
63%, respectively for cancers diagnosed between 1995-2000.
Therefore, treatments in addition to the standard methods of
treatment that include surgery, radiation, chemotherapy,
immunotherapy, and hormone therapy are needed.
[0005] Misregulation of genes that control cell fate determination
often contributes to cancer. Such altered genes are known as
oncogenes. Oncogenes are called proto-oncogenes when they are
normal (i.e., not mutated). Proto-oncogenes encode components of
the cell's normal growth-control pathway. Some of these components
are growth factors, receptors, signaling enzymes, and transcription
factors.
[0006] Ras is one such oncogene. Mammalian ras genes code for
closely related, small proteins (H-ras, K-ras and N-ras). Ras is
found in normal cells, where it helps to relay signals by acting as
a switch. When receptors on the cell surface are stimulated (by a
hormone, for example), Ras is switched on and transduces signals
that tell the cell to grow. If the cell-surface receptor is not
stimulated, Ras is not activated and so the pathway that results in
cell growth is not initiated. In about 30% of human cancers, Ras is
mutated so that it is permanently switched on, telling the cell to
grow regardless of whether receptors on the cell surface are
activated or not. A high incidence of ras gene mutations is found
in all lung cancers and adenocarcinomas (10% and 25%, respectively,
K-ras), in malignant tumors of the pancreas (80-90%, K-ras), in
colorectal carcinomas (30-60%, K-ras), in non-melanoma skin cancer
(30-50%, H-ras), in hematopoietic neoplasia of myeloid origin
(18-30%, K- and N-ras), and in seminoma (25-40%, K-ras). In other
tumors, a mutant ras gene is found at a lower frequency: for
example, in breast carcinoma (0-12%, K-ras), glioblastoma and
neuroblastoma (0-10%, K- and N-ras).
[0007] Other oncogenes include members of the MYC family (c-MYC,
NMYC, and L-MYC), which have been widely studied, and amplification
of myc genes has been found in a variety of tumor types including
lung (c-MYC, N-MYC, L-MYC), colon (c-MYC), breast (c-MYC), and
neuroblastoma (NMYC). Genes that inhibit apoptosis have also been
identified as oncogenes. The prototype of these genes is BCL-2.
Originally identified at the chromosomal breakpoint in follicular
lymphoma, this protein was found to inhibit cell death rather than
promote cell growth. BCL-2 belongs to a family of intracellular
proteins whose role is to regulate caspase activation that leads to
DNA fragmentation and cell death. In melanoma, BCL-2 has been
reported to be overexpressed in primary and metastatic lesions and
this phenotype is associated with tumor progression.
[0008] Radiation therapy is one of the three primary modalities
employed in cancer treatment. Although radiation has been in
practice for over a century, the global genetic response necessary
for tissues to survive radiation-induced injury remains largely
unknown. This has limited the ability to develop meaningful routes
to minimize normal tissue toxicity while enhancing tumor
eradication. While single-protein targeting strategies have shown
moderate success in preclinical models, few have been successful in
human trials. Ras overexpression in tumors is considered a poor
prognostic feature, and is hypothesized to be involved in the
response to cytotoxic therapy. Ras signaling has been shown to be
critical for protection from radiation induced target cell death
(Brown and Wilson, Canc. Biol. & Therapy, 2:477-490 (2003)).
Unfortunately, strategies directly targeting RAS or its upstream
and/or downstream effectors have not successfully altered the
radiation response in vivo.
[0009] A failure to identify radiation modulators may be due to the
complex genetic cellular response to radiation, as indicated by
microarray studies showing significant changes in the expression of
at least 855 genes (>1.5 fold) within 4 hours of radiation. This
suggests that regulatory molecules capable of regulating a large
number of target genes in a rapid manner may be required to affect
the radiation response.
[0010] Micro RNAs (referred to as "miRNAs") are small non-coding
RNAs, belonging to a class of regulatory molecules found in plants
and animals that control gene expression by binding to
complementary sites on target messenger RNA (mRNA) transcripts (SEQ
ID Nos. 1, 2 and 3) miRNAs are generated from large RNA precursors
(termed pri-miRNAs) that are processed in the nucleus into
approximately 70 nucleotide pre-miRNAs, which fold into imperfect
stem-loop structures (Lee, Y., et al., Nature (2003)
425(6956):415-9) (FIG. 1). The pre-miRNAs undergo an additional
processing step within the cytoplasm where mature miRNAs of 18-25
nucleotides in length are excised from one side of the pre-miRNA
hairpin by an RNase III enzyme, Dicer (Hutvagner, G., et al.,
Science (2001) 12:12 and Grishok, A., et al., Cell (2001)
106(1):23-34). mRNAs have been shown to regulate gene expression in
two ways. First, miRNAs that bind to protein-coding mRNA sequences
that are exactly complementary to the miRNA induce the RNA-mediated
interference (RNAi) pathway. Messenger RNA targets are cleaved by
ribonucleases in the RISC complex. This mechanism of miRNA-mediated
gene silencing has been observed mainly in plants (Hamilton, A. J.
and D. C. Baulcombe, Science (1999) 286(5441):950-2 and Reinhart,
B. J., et al., MicroRNAs in plants. Genes and Dev. (2002)
16:1616-1626), but an example is known from animals (Yekta, S., I.
H. Shih, and D. P. Bartel, Science (2004) 304(5670):594-6). In the
second mechanism, miRNAs that bind to imperfect complementary sites
on messenger RNA transcripts direct gene regulation at the
posttranscriptional level but do not cleave their mRNA targets.
mRNAs identified in both plants and animals use this mechanism to
exert translational control of their gene targets (Bartel, D. P.,
Cell (2004) 116(2):281-97).
[0011] Hundreds of miRNAs have been identified in the fly, worm,
plant and mammalian genomes. The biological role for the majority
of the miRNAs remains unknown because almost all of these were
found through cloning and bioinformatic approaches (Lagos-Quintana,
M., et al., Curr Biol (2002)12(9):735-9; Lagos-Quintana, M., et
al., RNA (2003) 9(2): 175-179; Lagos-Quintana, M., et al., Science
(2001) 294(5543): 853-8; Lee, R. C. and V. Ambros, Science (2001)
294(5543):862-4; Lau, N. C., et al., Science (2001)
294(5543):858-62; Lim, L. P., et al., Genes Dev (2003)
17(8):991-1008; Johnston, R. J. and O. Hobert, Nature (2003)
426(6968):845-9; and Chang, S., et al. Nature (2004)
430(7001):785-9).
[0012] It is likely that these uncharacterized miRNAs act as
important gene regulators during development to coordinate proper
organ formation, embryonic patterning, and body growth, but this
remains to be established. In zebrafish, most miRNAs are expressed
from organogenesis onward (Chen, P. Y., et al., Genes Dev (2005)
19(11):1288-93 and Wienholds, E., et al., Science, (2005)).
[0013] The biological roles for several miRNAs have been
elucidated. These studies highlight the importance of these
regulatory molecules in a variety of developmental and metabolic
processes. For example, the Drosophila miRNA, bantam, was
identified in a gain-of-function genetic screen for factors that
caused abnormal tissue growth (Brennecke, J., et al., Cell (2003)
113(1):25-36). Bantam was found to induce tissue growth in the fly
by both stimulating cell proliferation and inhibiting apoptosis
(Brennecke, J., et al., Cell (2003). 113(1):25-36). Although the
proliferation targets for bantam have not been identified, a
pro-apoptotic gene, hid, was shown to have multiple bantam
complementary sites in its 3'UTR. Since hid gene expression was
repressed by the bantam miRNA, this implicates a role for bantam in
controlling apoptosis by blocking hid function. Another Drosophila
miRNA, mir-14, was identified in a genetic screen for factors that
modified Reaper-induced apoptosis in the fly eye (Xu, P., et al.,
Curr Biol (2003) 13(9):790-5). mir-14 was shown to be a strong
suppressor of apoptosis. In addition, mir-14 also appears to play a
role in the Drosophila stress response as well as in regulating fat
metabolism. mRNAs also regulate Notch pathway genes in Drosophila
(Lai, et al. Genes Dev (2005) 19(9):1067-80). A mammalian miRNA,
mir-181, was shown to direct the differentiation of human B cells
(Chen, C. Z., et al., Science (2004) 303(5654): 83-6), mir-373
regulates insulin secretion (Poy, M. N., et al., Nature (2004)
432(7014):226-30), while other miRNAs regulate viral infections
(Lecellier, C. H., et al., Science (2005) 308(5721):557-60 and
Sullivan, C. S., et al., Nature (2005) 435(7042):682-6).
[0014] Studies to understand the mechanism of RNAi in C. elegans,
Drosophila and human cells have shown that the miRNA and RNAi
pathways may intersect (Grishok, A., et al. Cell (2001)
106(1):23-34 and Hutvagner, G., et al., Science (2001)
293(5531):834-8). mRNAs copurify with components of the RNAi
effector complex, RISC, suggesting a link between miRNAs and siRNAs
involved in RNAi (Mourelatos, Z., et al., Genes Dev (2002)
16(6):720-8; Hutvagner, G. and P. D. Zamore, Science (2002)
297(5589):2056-60; and Caudy, A. A., et al., Nature (2003)
425(6956): 411-4). There is also an indication that some protein
factors may play a role in both the miRNA ribonucleoprotein (miRNP)
and RISC and others might be unique to the miRNP (Grishok, A., et
al., Cell (2001) 106(1): 23-34 and Cannell, M. A., et al., Genes
Dev (2002) 16(21): 2733-42). For example, proteins of the
argonaute/PAZ/PIWI family are components of both RISC and miRNPs.
There is also mounting evidence that genes encoding these proteins
are linked to cancer. hAgo3, hAgo1, and hAgo4 reside in region
1p34-35, often lost in Wilms' tumors, and Hiwi, is located on
chromosome 12q24.33, which has been linked to the development of
testicular germ cell tumors (Carmell, M. A., et al., Genes Dev
(2002) 16(21):2733-42). In addition, DICER, the enzyme which
processes miRNAs and siRNAs, is poorly expressed in lung cancers
(Karube, Y., et al., Cancer Sci (2005) 96(2):111-5).
[0015] It is therefore an object of the present invention to
provide naturally occurring miRNAs for inhibition of expression of
one or more oncogenes.
[0016] It is further an object of the present invention to provide
naturally occurring nucleic acids for treatment or prophylaxis of
one or more symptoms of cancer.
[0017] It is an even further object of the present invention to
provide methods for sensitizing cancer cells to cytotoxic therapies
including radiotherapy and chemotherapy.
BRIEF SUMMARY OF THE INVENTION
[0018] Genes that control cell differentiation and development are
frequently mutated in human cancers. These include, but are not
limited to, oncogenes such as RAS, c-myc and bcl-2. Naturally
occurring microRNAs, in particular let-7, have been found that down
regulate these oncogenes in humans. Some of the let-7 genes are
located in chromosomal regions that are deleted in certain cancers.
Therefore, up-regulating these specific microRNAs or providing
analogous pharmaceutical compounds exogenously, should be effective
cancer therapies for tumors resulting from activation or
over-expression of these oncogenes. mRNAs nucleic acids including
pri-miRNA, pre-miRNA, mature miRNA or fragments of variants thereof
that retain the biological activity of the mature miRNA and DNA
encoding a pri-miRNA, pre-miRNA, mature miRNA, fragments or
variants thereof, or regulatory elements of the miRNA, referred to
jointly as "miRNAs" unless otherwise stated, are described. In one
embodiment, the size range of the miRNA can be from 21 nucleotides
to 170 nucleotides, although miRNAs of up to 2000 nucleotides can
be utilized. In a preferred embodiment the size range of the miRNA
is from 70 to 170 nucleotides in length. In another preferred
embodiment, mature miRNAs of from 21 to 25 nucleotides in length
can be used.
[0019] These miRNAs are useful as diagnostics and as therapeutics.
In one embodiment, the compositions are administered prior to
administration of radiotherapy to sensitize cells to the effects of
the radiation. The compositions are administered to a patient in
need of treatment of at least one symptom or manifestation (since
disease can occur/progress in the absence of symptoms) of cancer.
Aberrant expression of oncogenes is a hallmark of cancer, for
example, lung cancer. The compositions can be administered alone or
in combination with adjuvant cancer therapy such as surgery,
chemotherapy, radiotherapy, thermotherapy, immunotherapy, hormone
therapy and laser therapy, to provide a beneficial effect, e.g.
reduce tumor size, reduce cell proliferation of the tumor, inhibit
angiogenesis, inhibit metastasis, or otherwise improve at least one
symptom or manifestation of the disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 depicts the predicted secondary structure of the
let-7 pre-miRNA from various organisms (SEQ ID Nos. 1, 2 and 3).
The shaded residues indicate the mature miRNA transcript excised by
Dicer.
[0021] FIG. 2 is a schematic of potential RNA/RNA duplexes between
a C. elegans miRNA, let-7, (SEQ ID No. 6) and the target mRNA,
lin-41 (SEQ ID Nos. 4 and 5). The position of a loss-of-function
mutation for the let-7 miRNA, let-7(n2853), is shown by an arrow
below the duplexes.
[0022] FIG. 3 depicts let-7 homologues from various species (SEQ ID
Nos. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24 and 25), including humans and mouse. There are 2 human
homologues of lin-4 (SEQ ID No. 22), mir-125a (SEQ ID No. 25) and
mir-125b (SEQ ID No. 23). mir-237 (SEQ ID No. 24) is a C. elegans
lin-4 homologue.
[0023] FIG. 4A depicts potential LCSs in C. elegans let-60/RAS mRNA
3'UTR, black arrows indicate sites with similarity between C.
elegans and C. briggsae and white arrows indicate non-similar
sites. Shown below are predicted duplexes formed by LCSs (SEQ ID
Nos. 26, 27, 28, 29, 32, 33, 34 and 35) (top) and miR-84 (SEQ ID
Nos. 30 and 31) (bottom). let-7 and miR-84 are so similar that most
let-7 sites are also potential miR-84 sites.
[0024] FIG. 4B-depicts that the H.s. NRAS mRNA 3'UTR has 9 (SEQ ID
Nos. 36, 37, 38, 40, 41, 42, 43, 44 and 45). Black arrows indicate
sites conserved among mammalian species (in most cases human, rat,
mouse, hamster and guinea pig). Shown below are hypothesized
duplexes formed by (top) and let-7a miRNA (SEQ ID No. 39)
(bottom).
[0025] FIG. 4C depicts that the H.s. KRAS mRNA 3'UTR has 8 (SEQ ID
Nos. 46, 47, 48, 49, 50, 51, 52 and 53) potential LCSs. Black
arrows indicate sites conserved among mammalian species (in most
cases human, rat, mouse, hamster and guinea pig). Shown below are
hypothesized duplexes formed by (top) and let-7a miRNA (SEQ ID No.
39) (bottom).
[0026] FIG. 4D depicts that the H.s. HRAS mRNA 3'UTR has 3 (SEQ ID
Nos. 54, 55 and 56) potential LCSs. Black arrows indicate sites
conserved among mammalian species (in most cases human, rat, mouse,
hamster and guinea pig). Shown below are hypothesized duplexes
formed by (top) and let-7a miRNA (SEQ ID No. 39) (bottom).
[0027] FIG. 5A is a graph of the quantitative analysis of the
expression pattern from five independent wild-type transgenic lines
grown at 20.degree. C. At least 25% repression was observed in all
lines. A non-regulated lin-41 3'UTR missing its LCSs (pFS1031),
tested in duplicate is shown as a control.
[0028] FIG. 5B is a graph depicting the down-regulation of the
reporter gene expression is lost in let-7(n2853) mutant worms grown
at the permissive temperature, 15.degree. C. The parental (N2) line
was tested in triplicate; four isogenic let-7(n2853) mutant lines
were tested. Error bars represent standard deviations.
[0029] FIGS. 6A-C are graphs of the quantification of expression
data performed in triplicate. gj354 is a fusion of gA' to the
unc-54 3'UTR, driven by the lin-3 1 promoter. Error bars represent
standard deviations.
[0030] FIG. 7A is a graph of the quantification of the RAS antibody
fluorescence from replicates of the transfections. HEPG2 cells were
transfected with 10 and 30 nM of a let-7 or negative control
precursor miRNA. Immunofluorescence using an antibody specific to
NRAS, VRAS, and KRAS revealed that the let-7 transfected cells have
much lower levels of the RAS proteins. Thus, the presence of let-7
influences the expression of RAS in human cells.
[0031] FIG. 7B is a graph of the quantification of the fold
induction of RAS protein levels from replicates of the
transfections. HeLa cells were transfected with 100 nM let-7
inhibitor or negative control inhibitor. RAS immunofluorescence
revealed that cells transfected with the let-7 inhibitor has
increased levels of the RAS proteins relative to the negative
control transfected cells.
[0032] FIG. 8A is a schematic showing the NRAS short (NRAS S), NRAS
long (NRAS L) and KRAS 3'UTRs. Arrows indicate LCSs. The blackened
areas indicate the sequence cloned behind the reporter.
[0033] FIG. 8B is a graph of the relative repression of firefly
luciferase expression standardized to a transfection control,
renilla luciferase. pGL3-Cont is the empty vector.
[0034] FIG. 8C is a graph of the induction of firefly luciferase
expression when reporter plasmids with 3'UTR domains corresponding
to KRAS and NRAS are co-transfected with an inhibitor of let-7,
relative to a control inhibitor. Thus, the 3'UTRs of NRAS and KRAS
enable let-7 regulation.
[0035] FIG. 9A is a graph of the expression of let-7 in 21 breast,
colon, and lung tumors relative to associated normal adjacent
tissue (NAT). Fluorescently labeled miRNA was hybridized to
microarrays that included probes specific to let-7a and let-7c.
Fluorescence intensities for the tumor and NAT were normalized by
total fluorescence signal for all elements and the relative average
signal from the let-7 probes in the tumor and normal adjacent
samples are expressed as log ratios.
[0036] FIG. 9B is a graph of the correlation between RAS protein
and let-7c expression in tumor and normal adjacent tissue samples
from three lung squamous cell carcinomas. GAPDH and RAS proteins
were measured from crude extracts of tumor and normal adjacent
tissues using western analysis. The two proteins were assessed
simultaneously by mixing the antibodies used for detection. The
small RNA northern blot was assayed sequentially with radio-labeled
probes specific to let-7c and U6 snRNA. NRAS mRNA in the tumor and
normal adjacent tissues samples was measured by real-time PCR. The
real-time data were normalized based on the real-time PCR detection
of 18S rRNA in the various samples. The relative expression of NRAS
in the normal adjacent tissues was taken to be 100% and the Ct
value of NRAS in the tumor samples was used to assign the relative
expression of NRAS in the tumor samples.
[0037] FIG. 10A is a sequence comparison of the let-7 family of
miRNAs (SEQ ID Nos. 19, 21, 58 and 59) in C. elegans. The Majority
sequence is represented by SEQ ID No. 57.
[0038] FIG. 10B is a dendrogram of let-7 family members.
[0039] FIG. 11 depicts potential LCSs (SEQ ID Nos. 60, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and
80) in let-60/RAS in C. 30 elegans mRNA.
[0040] FIG. 12 is a sequence alignment of C. elegans (SEQ ID No.
81) (top) and C. briggsae (SEQ ID No. 82) (bottom) let-60/RAS mRNAs
(consensus sequence shown in middle) (SEQ ID No. 83). LCSs are
shown as black boxes.
[0041] FIG. 13A is a sequence alignment of C. elegans LET-60 (SEQ
ID No. 87) with other RAS and RAS related proteins from humans (SEQ
ID Nos. 84, 85 and 86) and C. elegans (SEQ ID Nos. 88, 89, 90 and
91).
[0042] FIG. 13B is a dendrogram showing the relationship of LET-60
to other RAS related proteins.
[0043] FIG. 13C is a graph showing quantification of the partial
suppression of let-60(gf) alleles. o84-X are lines overexpressing
mir-84, and TOPO--X are lines with the empty vector control. The
average of each of the three experimental and control lines is
indicated. The alignment from FIG. 13A, the dendrogram from FIG.
13B, and this graph demonstrate that Let-60 is the ortholog of
human HRAS, KRAS, and NRAS proteins.
[0044] FIG. 14 is a sequence alignment of partial sequences from
rodent (SEQ ID Nos. 94, 95 and 96) and human (SEQ ID Nos. 92 and
93) NRAS 3'UTRs. The LCSs shown in this alignment are boxed in
black.
[0045] FIG. 15 (SEQ ID Nos. 39, 97, 98, 99, 100, 101, 102 and 103)
depicts potential let-7::LCS duplexes formed with Xenopus laevis
and Danio rerio NRAS 3'UTRs.
[0046] FIG. 16A is a graph of the quantification of the antibody
fluorescence from replicates of HepG2 cells transfected with let-7
or negative control siRNAs using antibodies specific to GAPDH or
p21.
[0047] FIG. 16B is a graph of the quantification of fluorescent
signal from a single field of 50-100 cells for both the NRAS- and
Negative Control siRNA transfections. The data from this graph and
the graph shown in FIG. 16A demonstrate that the presence of let-7
does not influence the expression of control proteins in human
cells.
[0048] FIG. 17 is a graph of the expression of let-7c and let-7g in
lung tumors relative to associated normal adjacent tissue
(NAT).
[0049] FIG. 18A is a graph of the inhibition of let-7 that results
in a 100% increase of A549 cell numbers, compared to a control
transfection and a control anti-miRNA (mir-19a).
[0050] FIG. 18B is a graph of extra let-7 that causes a decrease in
A549 cell numbers compared to a control miRNA (NC).
[0051] FIG. 19 depicts potential let-7 complementary sites (boxed)
in the 3'UTR of human MYC (SEQ ID No. 104) and other vertebrates
(SEQ ID Nos. 105, 106 and 107) and potential duplexes between let-7
(SEQ ID No. [[99]] 39 and 110) and human MYC (SEQ ID Nos. 108 and
109).
[0052] FIG. 20A is a graph of the reduced expression of MYC and
BCL-2 protein in cells treated with exogenous let-7 miRNA compared
to a control miRNA with a scrambled let-7 sequence.
[0053] FIG. 20B is a graph of the increased expression of MYC and
BCL-2 in HeLa cells transfected with an anti-let-7 molecule.
[0054] FIG. 21A is a line graph showing the levels (Log2 Ratio) of
the indicated let-7 microRNAs (hsa-let-7a .diamond-solid.;
hsa-let-7b .box-solid.; hsa-let-7c .tangle-solidup.; hsa-let7d x;
hsa-let-7e *; hsa-let-7f ; hsa-let-7g+; hsa-let-7i ) collected from
cells prior to, and at 2, 8 and 24 hours post irradiation (2.5
Gray). The ratio of let-7 levels is shown with the unirradiated 0
time point as a baseline and error bars represent standard
deviation. Specifically, this graph shows the results obtained from
RNA isolated from A549 cells.
[0055] FIG. 21B is a line graph showing the levels (Log2 Ratio) of
the indicated let-7 microRNAs (hsa-let-7a .diamond-solid.;
hsa-let-7b .box-solid.; hsa-let-7c .tangle-solidup.; hsa-let7d x;
hsa-let-7e *; hsa-let-7f ; hsa-let-7g+; hsa-let-7i ) collected from
cells prior to, and at 2, 8 and 24 hours post irradiation (2.5
Gray). The ratio of let-7 levels is shown with the unirradiated 0
time point as a baseline and error bars represent standard
deviation. Specifically, this graph shows the results obtained from
RNA isolated from CRL2741 lung epithelial cells.
[0056] FIG. 21C is a series of heat maps demonstrating let-7
changes post-irradiation in A549 cells and CRL2741 cells with
similar changes in let-7 ratios observed over the first twenty-four
hours post-radiation.
[0057] FIG. 22A is a line graph showing the survival (proportion
survival) of A549 cells transfected with let-7b and irradiated 24
hours later with 2.0, 4.0 or 6.0 Grays (Gy). Survival was measured
using a clonogenic assay. Error bars represent standard
deviation.
[0058] FIG. 22B is a line graph showing the survival (proportion
survival) of A549 cells transfected with let-7g and irradiated 24
hours later with 2.0, 4.0 or 6.0 Grays (Gy). Survival was measured
using a clonogenic assay. Error bars represent standard
deviation.
[0059] FIG. 22C is a line graph showing the survival (proportion
survival) of A549 cells transfected with anti-let-7b and irradiated
24 hours later with 2.0, 4.0 or 6.0 Grays (Gy). Survival was
measured using a clonogenic assay. Error bars represent standard
deviation.
[0060] FIG. 22D is a line graph showing the survival (proportion
survival) of A549 cells transfected with anti-let-7g and irradiated
24 hours later with 2.0, 4.0 or 6.0 Grays (Gy). Survival was
measured using a clonogenic assay. Error bars represent standard
deviation.
[0061] FIG. 23A is a line graph showing the effects of
overexpression of let-7 and its homologue mir-84 on
radiosensitization in the Radelegans C. elegans radiation model.
Radiosensitization is measured as a percent of wild-type vulvae in
wild-type (.diamond-solid.) let-7 overexpressing (.box-solid.) or
mir-84 overexpressing (.tangle-solidup.) C. elegans treated with
the indicated doses of radiation (Gy). P-values are listed next to
the curves they represent compared to wild-type animals.
[0062] FIG. 23B is a line graph showing the effects of loss of
mir-84 expression on radiosensitization in the Radelegans C.
elegans radiation model. mir-84 deletion mutants mir-84(tm1304)
(.box-solid.) were compared to wild type (.diamond-solid.) at the
indicated doses of radiation (Gy). Radiosensitization is measured
as a percent of wild-type vulvae in wild-type. The effect of RNAi
against the C. elegans RAS homologue using let-60/RAS (RNAi) in a
mir-84 deletion background (.tangle-solidup.) is also shown. P
value represents the mir-84 deletion strain compared to wild-type
animals.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0063] As used herein the term "nucleic acid" refers to multiple
nucleotides (i.e. molecules comprising a sugar (e.g. ribose or
deoxyribose) linked to a phosphate group and to an exchangeable
organic base, which is either a substituted pyrimidine (e.g.
cytosine (C), thymidine (T) or uracil (U)) or a substituted purine
(e.g. adenine (A) or guanine (G)). The term shall also include
polynucleosides (i.e. a polynucleotide minus the phosphate) and any
other organic base containing polymer. Purines and pyrimidines
include but are not limited to adenine, cytosine, guanine,
thymidine, inosine, 5-methylcytosine, 2-aminopurine,
2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other
naturally and non-naturally occurring nucleobases, substituted and
unsubstituted aromatic moieties. Other such modifications are well
known to those of skill in the art. Thus, the term nucleic acid
also encompasses nucleic acids with substitutions or modifications,
such as in the bases and/or sugars.
[0064] As used herein, the term "microRNA" refers to any type of
interfering RNA, including but not limited to, endogenous microRNA
and artificial microRNA. Endogenous microRNA are small RNAs
naturally present in the genome which are capable of modulating the
productive utilization of mRNA. The term artificial microRNA
includes any type of RNA sequence, other than endogenous microRNA,
which is capable of modulating the productive utilization of
mRNA.
[0065] "MicroRNA flanking sequence" as used herein refers to
nucleotide sequences including microRNA processing elements.
MicroRNA processing elements are the minimal nucleic acid sequences
which contribute to the production of mature microRNA from
precursor microRNA. Precursor miRNA termed pri-miRNAs are processed
in the nucleus into about 70 nucleotide pre-miRNAs, which fold into
imperfect stem-loop structures. The microRNA flanking sequences may
be native microRNA flanking sequences or artificial microRNA
flanking sequences. A native microRNA flanking sequence is a
nucleotide sequence that is ordinarily associated in naturally
existing systems with microRNA sequences, i.e., these sequences are
found within the genomic sequences surrounding the minimal microRNA
hairpin in vivo. Artificial microRNA flanking sequences are
nucleotides sequences that are not found to be flanking to microRNA
sequences in naturally existing systems. The artificial microRNA
flanking sequences may be flanking sequences found naturally in the
context of other microRNA sequences. Alternatively they may be
composed of minimal microRNA processing elements which are found
within naturally occurring flanking sequences and inserted into
other random nucleic acid sequences that do not naturally occur as
flanking sequences or only partially occur as natural flanking
sequences.
[0066] The microRNA flanking sequences within the precursor
microRNA molecule may flank one or both sides of the stem-loop
structure encompassing the microRNA sequence. Preferred structures
have flanking sequences on both ends of the stem-loop structure.
The flanking sequences may be directly adjacent to one or both ends
of the stem-loop structure or may be connected to the stem-loop
structure through a linker, additional nucleotides or other
molecules.
[0067] As used herein a "stem-loop structure" refers to a nucleic
acid having a secondary structure that includes a region of
nucleotides which are known or predicted to form a double strand
(stem portion) that is linked on one side by a region of
predominantly single-stranded nucleotides (loop portion). The terms
"hairpin" and "fold-back" structures are also used herein to refer
to stem-loop structures. Such structures and terms are well known
in the art. The actual primary sequence of nucleotides within the
stem-loop structure is not critical as long as the secondary
structure is present. As is known in the art, the secondary
structure does not require exact base-pairing. Thus, the stem may
include one or more base mismatches. Alternatively, the
base-pairing may not include any mismatches.
[0068] As used herein, the term "let-7" refers to the nucleic acid
encoding the let-7 miRNA and homologues and variants thereof
including conservative substitutions, additions, and deletions
therein not adversely affecting the structure or function.
Preferably, let-7 refers to the nucleic acid encoding let-7 from C.
elegans (NCBI Accession No. AY390762), most preferably, let-7
refers to the nucleic acid encoding a let-7 family member from
humans, including but not limited to, NCBI Accession Nos. AJ421724,
A.1421725, AJ421726, AJ421727, AJ421728, AJ421729, AJ421730,
AJ421731, AJ421732, and biologically active sequence variants of
let-7, including alleles, and in vitro generated derivatives of
let-7 that demonstrate let-7 activity.
[0069] Sequence variants of let-7 fall into one or more of three
classes: substitutional, insertional or deletional variants.
Insertions include 5' and/or 3' terminal fusions as well as
intrasequence insertions of single or multiple residues. Insertions
can also be introduced within the mature sequence of let-7. These,
however, ordinarily will be smaller insertions than those at the 5'
or 3' terminus, on the order of 1 to 4 residues.
[0070] Insertional sequence variants of let-7 are those in which
one or more residues are introduced into a predetermined site in
the target let-7. Most commonly insertional variants are fusions of
nucleic acids at the 5' or 3' terminus of let-7. Deletion variants
are characterized by the removal of one or more residues from the
let-7 RNA sequence. These variants ordinarily are prepared by site
specific mutagenesis of nucleotides in the DNA encoding let-7,
thereby producing DNA encoding the variant, and thereafter
expressing the DNA in recombinant cell culture. However, variant
let-7 fragments may be conveniently prepared by in vitro synthesis.
The variants typically exhibit the same qualitative biological
activity as the naturally-occurring analogue, although variants
also are selected in order to modify the characteristics of
let-7.
[0071] Substitutional variants are those in which at least one
residue sequence has been removed and a different residue inserted
in its place. While the site for introducing a sequence variation
is predetermined, the mutation per se need not be predetermined.
For example, in order to optimize the performance of a mutation at
a given site, random mutagenesis may be conducted at the target
region and the expressed let-7 variants screened for the optimal
combination of desired activity. Techniques for making substitution
mutations at predetermined sites in DNA having a known sequence are
well known.
[0072] Nucleotide substitutions are typically of single residues;
insertions usually will be on the order of about from 1 to 10
residues; and deletions will range about from 1 to 30 residues.
Deletions or insertions preferably are made in adjacent pairs; i.e.
a deletion of 2 residues or insertion of 2 residues. Substitutions,
deletion, insertions or any combination thereof may be combined to
arrive at a final construct. Changes may be made to increase the
activity of the miRNA, to increase its biological stability or
half-life. All such modifications to the nucleotide sequences
encoding such miRNA are encompassed.
[0073] A DNA isolate is understood to mean chemically synthesized
DNA, cDNA or genomic DNA with or without the 3' and/or 5' flanking
regions. DNA encoding let-7 can be obtained from other sources by
a) obtaining a cDNA library from cells containing mRNA, b)
conducting hybridization analysis with labeled DNA encoding let-7
or fragments thereof (usually, greater than 100 bp) in order to
detect clones in the cDNA library containing homologous sequences,
and c) analyzing the clones by restriction enzyme analysis and
nucleic acid sequencing to identify full-length clones.
[0074] As used herein nucleic acids and/or nucleic acid sequences
are homologous when they are derived, naturally or artificially,
from a common ancestral nucleic acid or nucleic acid sequence.
Homology is generally inferred from sequence similarity between two
or more nucleic acids or proteins (or sequences thereof). The
precise percentage of similarity between sequences that is useful
in establishing homology varies with the nucleic acid and protein
at issue, but as little as 25% sequence similarity is routinely
used to establish homology. Higher levels of sequence similarity,
e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more can
also be used to establish homology. Methods for determining
sequence similarity percentages (e.g., BLASTN using default
parameters) are generally available. Software for performing BLAST
analyses is publicly available through the National Center for
Biotechnology Information (www.ncbi.nlm.nih.gov).
I. Compositions
[0075] Genes that control cell differentiation and development are
frequently mutated in human cancers. These include, but are not
limited to, oncogenes such as RAS, c-myc and bc1-2. Naturally
occurring microRNAs, in particular let-7, have been found that down
regulate these oncogenes in humans. Some of the let-7 genes are
located in chromosomal regions that are deleted in certain cancers.
Therefore, up-regulating these specific microRNAs or providing
analogous pharmaceutical compounds exogenously, should be effective
cancer therapies for tumors resulting from activation or
over-expression of these oncogenes.
[0076] In preferred embodiments, the miRNA formulations are
administered to individuals with a cancer that expresses one or
more targets of let-7 or lin-4. More preferably, the formulations
are administered to individuals with a cancer that over expresses
RAS, MYC and/or BCL-2 or other target having one or more binding
sites for the let-7. Multiple different pathways, along with the
RAS/MAPK pathway, are implicated in cancer. The 3'UTRs of many
known cancer genes have been examined and potential let-7
complementary sites have been identified in the 3'UTR of many of
them (FIG. 19 and Table 1). These sites possess features of
established let-7 complementary sites in known C. elegans let-7
targets (Reinhart, et al., Nature (2000) 403:901-906; Johnson, et
al., Cell (2005) 120(5):635-47; Grosshans, et al., Dev Cell (2005)
8(3):321-30; Lin, et al., Dev Cell (2003) 4(5):639-50; Slack, et
al., Molec. Cell (2000) 5:659669; Vella, et al., Genes Dev (2004)
18(2):132-7). let-7 complementary sites (LCS) in the 3'Ul'R5 of
human c-MYC and BCL-2 have been identified (see FIG. 19 for c-MYC).
Many of the potential target genes shown in Table 1 are also
up-regulated in lung cancer as well as other cancers, leading to
the conclusion that let-7 is responsible for repressing their
expression in normal tissues. Some genes, like GRB2, have a similar
number of LCSs as known let-7 target genes like KRAS (Table 1).
VEGF was also identified as having let-7 complementary sites. These
results indicate that let-7 can repress expression of multiple
oncogenes. In addition to inhibiting cell proliferation, let-7 may
also inhibit angiogenesis. Therefore, administration of let-7 may
inhibit multiple pathways that promote survival of cancer or tumor
cells (i.e., angiogenesis, decreased apoptosis and increased cell
proliferation).
TABLE-US-00001 TABLE 1 Genes implicated in cancer that contain
let-7 binding sites. Homo Sapiens Gene Number of LCS sites EGF 1
EGFR 1 ERBB3 3 GRB2 10 NRAS 9 KRAS2 8 HRAS 3 RAF1 1 ARAF 3 MAP2K2 2
MAPK1 1 MAPK3 4 MET 3 KIT 3 TP73L(AIX) 5 MYC 2 MYCL1 6 MYCN 4 BCL2
5 BCL2L1 5 BCL2L2 6 CCND1/cyclinD 3 CDK4 1 MDM2/HDM2 4 FES 2 FURIN
2 INSL3 2 CSF1R/FMS 1 MYBL2 1 MYB 1 PIK3CD 6 PIK3C2B 4 PIK3CG 1
PIK3R5 1 TERT 3 AKT1 3 AKT3 2 VEGF 3 HLIN-41 4 VDR 7 PXR 3 FOXA1 2
FOXA2 A ASH1L 2 ARID1B 5 GR 2 GLI2 1 14-3-3zeta 6 MO25 1 SMG1 2
FRAP1 3 PER2 4
miRNAs Have Known Roles in Human Cancer
[0077] Recent studies from different laboratories show roles for
miRNAs in human cancer (McManus, Seminars in Cancer Biology (2003)
13:253-258). The human miRNAs, mir-15 and mir-16, are
preferentially deleted or down-regulated in patients with a common
form of adult leukemia, B cell chronic lymphocytic leukemia (Cahn,
et al., Proc Natl Acad Sci USA (2002) 99(24):15524-9). This study
suggests that miRNAs may function as tumor suppressor genes. The
bic locus, which encodes the mir-155 miRNA works cooperatively with
c-myc and induces B-cell lymphomas, presumably acting as a
proto-oncogene (Haasch, D., et al., Cell Immunol (2002)
217(1-2):78-86). Mir-142 acts as a tumor suppressor in chronic
lymphocytic leukemia (Cahn, G. A., et al., Proc Natl Acad Sci USA
(2002) 99(24):15524-9; Lagos-Quintana, M., et al., Curr Biol (2002)
12(9):735-9). His-1 acts as an oncogene in B cell lymphoma (Haasch,
D., et al., (2002); Lagos-Quintana, M., et al., (2002);
Lagos-Quintana, M., et al., Science (2001) 294(5543):853-8; L1, et
al. Am J Pathol (1997) 150:1297-305). Translocation of myc to the
mir-142 locus causes B cell lymphoma (Lagos-Quintana, M., et al.,
(2002); Gauwerky, C. E., et al., Proc Natl Acad Sci USA, 1989.
86(22):8867-71). Mir-143 and mir-145 are poorly expressed in
colorectal cancer (Michael, M. Z., et al., Mol Cancer Res (2003)
1(12):882-91). Over-expression of the mir-17, 18, 19, 20 locus is
able to cause lymphomas in a mouse model and is up-regulated by MYC
(He, L., et al, Nature (2005) 435(7043):828-33; O'Donnell, K. A.,
et al., Nature (2005) 435(7043):839-43).
[0078] Misregulation of genes that control cell fate determination
often contributes to cancer. Genes that control cell
differentiation and development are frequently mutated in human
cancer. The model organism Caenorhabditis elegans has been used to
identify genes required for cell differentiation in a stem-cell
like lineage in the epidermis. C. elegans growth and development is
divided into three major stages called embryo, larva and adult.
Larval growth is subdivided into four larval stages (L1, L2, L3 and
L4). Each larval stage ends in a molt and ultimately the animal
matures into an adult. The genes that regulate timing of
stage-appropriate cell division and differentiation are known as
heterochronic genes (Slack, F. and G. Ruvkun, Annu Rev Genet (1997)
31:611-34; Banerjee, D. and F. Slack, Bioessays (2002)
24(2):119-29). In C. elegans, heterochronic genes control the
timing of cell fate determination and differentiation. In
heterochronic mutants, cells frequently fail to terminally
differentiate, and instead divide again, a hallmark of cancer.
[0079] The founding members of the miRNA family, lineage
defective-4 (lin-4) and lethal-7 (let-7), were identified through
genetic analysis to control the timing of stage-appropriate cell
division and differentiation in C. elegans (Lee, et al. Cell (1993)
75(5):843-854; Reinhart, B., et al., Nature (2000) 403: 901906;
Slack, F. and G. Ruvkun, Annu Rev Genet (1997) 31: 611-34;
Banerjee, D. and F. Slack, Bioessays (2002) 24(2):119-29). let-7
and lin-4 control the timing of proliferation versus
differentiation decisions. Some of these genes, like lin-4 and
let-7, encode microRNAs (miRNAs) that are conserved in humans.
Mutations in the lin-4 and let-7 miRNAs result in inappropriate
reiterations of the first larval stage (L1) and the fourth larval
stage (L4) fates, respectively, and these defects lead to
disruptions in cell cycle exit (Lee, et al. Cell (1993)
75(5):843-854; Reinhart, B., et al., Nature (2000) 403:901-906).
For example, in wild-type animals, specialized skin cells, known as
seam cells, divide with a stem cell pattern and terminally
differentiate at the beginning of the adult stage. The seam cells
fail to terminally differentiate in lin-4 and let-7 mutant animals,
and instead reiterate the larval fate and divide again. Lack of
cell cycle control and failure to terminally differentiate are
hallmarks of cancer.
[0080] The expression patterns for lin-4 and let-7 correlate with
their role in directing developmental timing lin-4 RNA accumulates
during the L1 stage and is responsible for the L1/L2 transition in
nematodes by inhibiting the expression of lin-14 and lin-28,
repressors of post-L1 fates (Lee, et al. Cell (1993) 75(5):843-854;
Ambros, V. and H. R. Horvitz, Science (1984) 226:409-416; Wightman,
et al. Cell (1993) 75(5):855-862; Moss, et al. Cell (1997) 88(5):
37-46; and Feinbaum, R. and V. Ambros, Dev Biol (1999)
210(1):87-95). let-7 RNA accumulates during the L4 stage and is
responsible for the L4/Adult transition by down-regulating the
expression of lin-41, hbl-1 and RAS (Johnson, et al., Cell (2005)
120(5):635-47; Grosshans, et al., Dev Cell (2005) 8(3)321-30; Lin,
et al., Dev Cell (2003) 4(5):639-50; Slack, F. J., Molec. Cell
(2000) 5:659669).
[0081] These 21-22 nucleotide miRNAs exert their effect by binding
to imperfect complementary sites within the 3'-untranslated regions
(3'UTRs) of their target protein-coding mRNAs and repress the
expression of these genes at the level of translation (Lee, et al.
Cell (1993) 75(5):843-854; Reinhart, B., et al., Nature (2000)
403:901-906; Moss, et al. Cell (1997) 88(5):637-46; Lin, S. Y., et
al., Dev Cell (2003) 4(5):639-50; Slack, F. J., et al., Molec. Cell
(2000) 5:659-669; Abrahante, J. E., et al., Dev Cell (2003)
4(5):625-37; and Olsen, P. H. and V. Ambros, Dev Biol (1999)
216(2):671-80). Deletion of let-7 miRNA complementary sites (LCS)
(SEQ ID No. 4 and SEQ ID No. 5) from the lin-41 3'UTR (FIG. 2)
showed abrogation of the normal down-regulation of lin-41 during
the L4 and adult stages and recent work has shown that these
complementary sites alone are sufficient for regulation on lin-41
(Reinhart, B., et al., Nature (2000) 403:901-906; Slack, F. J., et
al., Molec. Cell (2000) 5:659669; Vella, M. C., et al., Genes Dev
(2004) 18(2):132-7).
[0082] The let-7 target gene, lin-41 is similar to known oncogenes.
C. elegans lin-41 loss-of-function (1f) mutations cause cells to
terminally differentiate precociously, opposite [[to]] of the
effect seen with let-7(1f), while over-expression of lin-41 causes
let-7(1f)-like seam cell proliferation (Reinhart, B., et al.,
Nature (2000) 403: 901-906; Slack, F. J., et al., Molec. Cell
(2000) 5:659-669). Like let-7, lin-41 is an important cell
proliferation and differentiation gene. let-7 and lin-41 work
together to cause cells to proliferate or differentiate at the
right time.
[0083] lin-41 encodes a member of the RBCC (RING finger, B box,
Coiled Coil (Freemont, Ann. New York. Acad. Sci. (1993)
684:174-192) family of proteins. Members of this family have
diverse proposed functions, such as transcription and RNA binding,
and include the PML (Kakizuka, A., et al., Cell (1991) 66:663-674),
TIF1 (Le Dourarin, B., et al., EMBO J. (1995) 14(9):2020-2033) and
Rfp proto-oncogenes. The most common form of promyelocytic leukemia
involves a translocation that fuses PML to the RARIa gene. The
N-terminal part of TIF1, is fused to B-raf in the oncogenic protein
T18 (Le Dourarin, B., et al., EMBO J. (1995) 14(9):2020-2033).
Emu-ret mice, carrying an RFP/RET fusion gene under the
transcriptional control of the immunoglobulin heavy chain enhancer,
develop B lineage leukemias and lymphomas (Wasserman, et al. Blood
(1998) 92(1):273-82). In transformed NIH 3T3 cells, the
amino-terminal half of Rfp with a RING finger motif is fused to a
truncated Ret receptor tyrosine kinase, Rfp/Ret (Hasegawa, N., et
al., Biochem Biophys Res Commun (1996) 225(2):627-31). Members of
this family are associated with cancer progression. It is expected
that mammalian lin-41 is also a proto-oncogene.
[0084] In addition to lin-41, let-7 regulates other target genes in
a 3' UTR dependent manner, including hunchback-like 1 (hbl-1) (Lin
Shin-Yi, J., et al. Dev. Cell, 2003(4):1-20) and let-60, the C.
elegans RAS oncogene homologue (see Example 1). As shown in Table
2, let-60/RAS contains multiple let-7 complimentary sites in its
3'UTR and let-60 suppresses let-7 mutants. let-60/RAS is best
understood for its role in C. elegans vulval development and
let-60/RAS 3'UTR is sufficient to restrict let-60/RAS expression
only to the vulval precursor cell (VPC) that absolutely requires
let-60/ras activity (the primary induced cell, 1.degree. or P6.p
cell). In a normal animal, a let-7 family member, mir-84, is
expressed in all the VPCs except the primary induced cell, and
represses let-60/ras expression in these cells.
[0085] In animals carrying let-60 activating mutations, more than
one VPC is induced to differentiate into the 1.degree. cell fate,
leading to excess vulvae. Over-expression of mir-84 suppresses
activating mutations in let-60/RAS. Many activating mutations in
the human NRAS, KRAS and HRAS genes alter the same amino acid
affected by the C. elegans let-60 activating mutation. Since RAS is
mutated in multiple human cancers (Malumbres, et al. Nat Rev Cancer
(2003) 3(6):459-65), the hypothesis that human RAS is a target of
human let-7 was tested and determined to be (see example 1
below).
[0086] lin-4 and let-7 miRNAs are evolutionarily conserved in
higher animals, including humans (FIG. 3) (SEQ ID Nos. 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25) and
temporally expressed (see examples below) which implies a universal
role for these miRNAs during animal development (Lagos-Quintana,
M., et al., Mouse. Curr Biol (2002) 12(9):735-9 and Pasquinelli, A.
E., et al., Nature (2000) 408(6808):86-9). let-7 orthologues have
been identified in mammals, including humans, and let-7 is
expressed in human lung tissues as judged by northern blot
(Pasquinelli, A. E., et al.). There are 3 exact copies of the
mature let-7 sequence in the sequenced human genome (referred to as
let-7a1, let-7a2, let-7a3 under control of separate promoters) and
a variety of close homologues that differ from let-7 at certain
nucleotide positions (e.g. let-7c (SEQ ID No. 16), see FIG. 3). The
nematode, fly and human let-7 genes are processed from a precursor
form (pre-let-7) that is predicted to form a stem loop structure
which is also conserved (FIG. 1). Similarly, there are 2 human and
mouse homologues of lin-4 (SEQ ID No. 22), named mir-125a (SEQ ID
no. 25) and mir-125b (SEQ ID No. 23) (FIG. 3).
[0087] Recent work has demonstrated that microRNA expression
profiles can accurately diagnose particular cancers better than
standard messenger RNA expression profiles (Lu, et al., Nature
435:834-838 (2005)).
miRNAs Useful to Regulate Human Oncogenes
[0088] Naturally occurring microRNAs that regulate human oncogenes,
primiRNA, pre-miRNA, mature miRNA or fragments of variants thereof
that retain the biological activity of the mature miRNA and DNA
encoding a pri-miRNA, pre-miRNA, mature miRNA, fragments or
variants thereof, or regulatory elements of the miRNA, have been
identified. The size of the miRNA is typically from 21 nucleotides
to 170 nucleotides, although nucleotides of up to 2000 nucleotides
can be utilized. In a preferred embodiment the size range of the
pre-miRNA is between 70 to 170 nucleotides in length and the mature
miRNA is between 21 and 25 nucleotides in length.
Nucleic Acids
General Techniques
[0089] General texts which describe molecular biological techniques
include Sambrook, Molecular Cloning: a Laboratory Manual (2nd ed.),
Vols. 1-3, Cold Spring Harbor Laboratory, (1989); Current Protocols
in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New
York (1997); Laboratory Techniques in Biochemistry and Molecular
Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and
Nucleic Acid Preparation, P. Tijssen, ed. Elsevier, N.Y. (1993);
Berger and Kimmel, Guide to Molecular Cloning Techniques Methods in
Enzymology volume 152 Academic Press, Inc., San Diego, Calif. These
texts describe mutagenesis, the use of vectors, promoters and many
other relevant topics related to, e.g., the generation and
expression of genes that encode let-7 or any other miRNA activity.
Techniques for isolation, purification and manipulation of nucleic
acids, genes, such as generating libraries, subcloning into
expression vectors, labeling probes, and DNA hybridization are also
described in the texts above and are well known to one of ordinary
skill in the art.
[0090] The nucleic acids, whether miRNA, DNA, cDNA, or genomic DNA,
or a variant thereof, may be isolated from a variety of sources or
may be synthesized in vitro. Nucleic acids as described herein can
be administered to or expressed in humans, transgenic animals,
transformed cells, in a transformed cell lysate, or in a partially
purified or a substantially pure form.
[0091] Nucleic acids are detected and quantified in accordance with
any of a number of general means well known to those of skill in
the art. These include, for example, analytical biochemical methods
such as spectrophotometry, radiography, electrophoresis, capillary
electrophoresis, high performance liquid chromatography (HPLC),
thin layer chromatography (TLC), and hyperdiffusion chromatography,
various immunological methods, such as fluid or gel precipitin
reactions, immunodiffusion (single or double),
immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked
immunosorbent assays (ELISAs), immuno-fluorescent assays, and the
like, Southern analysis, Northern analysis, Dot-blot analysis, gel
electrophoresis, RT-PCR, quantitative PCR, other nucleic acid or
target or signal amplification methods, radiolabeling,
scintillation counting, and affinity chromatography.
[0092] Various types of mutagenesis can be used, e.g., to modify a
nucleic acid encoding a gene with let-7 or other miRNA activity.
They include but are not limited to site-directed, random point
mutagenesis, homologous recombination (DNA shuffling), mutagenesis
using uracil containing templates, oligonucleotide-directed
mutagenesis, phosphorothioate-modified DNA mutagenesis, and
mutagenesis using gapped duplex DNA. Additional suitable methods
include point mismatch repair, mutagenesis using repair-deficient
host strains, restriction-selection and restriction-purification,
deletion mutagenesis, mutagenesis by total gene synthesis,
double-strand break repair. Mutagenesis, e.g., involving chimeric
constructs, are also included in the present invention. In one
embodiment, mutagenesis can be guided by known information of the
naturally occurring molecule or altered or mutated naturally
occurring molecule, e.g., sequence, sequence comparisons, physical
properties, crystal structure. Changes may be made to increase the
activity of the miRNA, to increase its biological stability or
half-life.
[0093] Comparative hybridization can be used to identify nucleic
acids encoding genes with let-7 or other miRNA activity, including
conservative variations of nucleic acids.
[0094] Nucleic acids "hybridize" when they associate, typically in
solution. Nucleic acids hybridize due to a variety of well
characterized physico-chemical forces, such as hydrogen bonding,
solvent exclusion, base stackinge. An extensive guide to the
hybridization of nucleic acids is found in Tijssen (1993)
Laboratory Techniques in Biochemistry and Molecular
Biology-Hybridization with Nucleic Acid Probes part I chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," (Elsevier, N.Y.), as well as in
Ausubel, supra. Hames and Higgins (1995) Gene Probes 1 IRL Press at
Oxford University Press, Oxford, England, (Hames and Higgins 1) and
Haines and Higgins (1995) Gene Probes 2 IRL Press at Oxford
University Press, Oxford, England (Haines and Higgins 2) provide
details on the synthesis, labeling, detection and quantification of
DNA and RNA, including oligonucleotides.
[0095] Nucleic acids which do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, e.g., when a copy of a nucleic acid is created using the
maximum codon degeneracy permitted by the genetic code.
[0096] Suitable nucleic acids for use in the methods described
herein include, but are not limited to, pri-miRNA, pre-miRNA,
mature miRNA or fragments of variants thereof that retain the
biological activity of the miRNA and DNA encoding a pri-miRNA,
pre-miRNA, mature miRNA, fragments or variants thereof, or DNA
encoding regulatory elements of the miRNA.
Viral Vectors
[0097] In one embodiment the nucleic acid encoding a miRNA molecule
is on a vector. These vectors include a sequence encoding a mature
microRNA and in vivo expression elements. In a preferred
embodiment, these vectors include a sequence encoding a pre-miRNA
and in vivo expression elements such that the pre-miRNA is
expressed and processed in vivo into a mature miRNA. In another
embodiment, these vectors include a sequence encoding the pri-miRNA
gene and in vivo expression elements. In this embodiment, the
primary transcript is first processed to produce the stem-loop
precursor miRNA molecule. The stem-loop precursor is then processed
to produce the mature microRNA.
[0098] Vectors include, but are not limited to, plasmids, cosmids,
phagemids, viruses, other vehicles derived from viral or bacterial
sources that have been manipulated by the insertion or
incorporation of the nucleic acid sequences for producing the
microRNA, and free nucleic acid fragments which can be attached to
these nucleic acid sequences. Viral and retroviral vectors are a
preferred type of vector and include, but are not limited to,
nucleic acid sequences from the following viruses: retroviruses,
such as: Moloney murine leukemia virus; Murine stem cell virus,
Harvey murine sarcoma virus; murine mammary tumor virus; Rous
sarcoma virus; adenovirus; adeno-associated virus; SV40-type
viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses;
herpes viruses; vaccinia viruses; polio viruses; and RNA viruses
such as any retrovirus. One of skill in the art can readily employ
other vectors known in the art.
[0099] Viral vectors are generally based on non-cytopathic
eukaryotic viruses in which non-essential genes have been replaced
with the nucleic acid sequence of interest. Non-cytopathic viruses
include retroviruses, the life cycle of which involves reverse
transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Retroviruses have been
approved for human gene therapy trials. Genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of nucleic acids in vivo. Standard
protocols for producing replication-deficient retroviruses
(including the steps of incorporation of exogenous genetic material
into a plasmid, transfection of a packaging cell lined with
plasmid, production of recombinant retroviruses by the packaging
cell line, collection of viral particles from tissue culture media,
and infection of the target cells with viral particles) are
provided in Kriegler, M., "Gene Transfer and Expression, A
Laboratory Manual," W.H. Freeman Co., New York (1990) and Murry, E.
J. Ed. "Methods in Molecular Biology," vol. 7, Humana Press, Inc.,
Cliffton, N.J. (1991).
Promoters
[0100] The "in vivo expression elements" are any regulatory
nucleotide sequence, such as a promoter sequence or
promoter-enhancer combination, which facilitates the efficient
expression of the nucleic acid to produce the microRNA. The in vivo
expression element may, for example, be a mammalian or viral
promoter, such as a constitutive or inducible promoter or a tissue
specific promoter. Examples of which are well known to one of
ordinary skill in the art. Constitutive mammalian promoters
include, but are not limited to, polymerase promoters as well as
the promoters for the following genes: hypoxanthine phosphoribosyl
transferase (HPTR), adenosine deaminase, pyruvate kinase, and
beta.-actin. Exemplary viral promoters which function
constitutively in eukaryotic cells include, but are not limited to,
promoters from the simian virus, papilloma virus, adenovirus, human
immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus,
the long terminal repeats (LTR) of moloney leukemia virus and other
retroviruses, and the thymidine kinase promoter of herpes simplex
virus. Other constitutive promoters are known to those of ordinary
skill in the art. Inducible promoters are expressed in the presence
of an inducing agent and include, but are not limited to,
metal-inducible promoters and steroid-regulated promoters. For
example, the metallothionein promoter is induced to promote
transcription in the presence of certain metal ions. Other
inducible promoters are known to those of ordinary skill in the
art.
[0101] Examples of tissue-specific promoters include, but are not
limited to, the promoter for creatine kinase, which has been used
to direct expression in muscle and cardiac tissue and
immunoglobulin heavy or light chain promoters for expression in B
cells. Other tissue specific promoters include the human smooth
muscle alpha-actin promoter.
[0102] Exemplary tissue-specific expression elements for the liver
include but are not limited to HMG-COA reductase promoter, sterol
regulatory element 1, phosphoenol pyruvate carboxy kinase (PEPCK)
promoter, human C-reactive protein (CRP) promoter, human
glucokinase promoter, cholesterol 7-alpha hydroylase (CYP-7)
promoter, beta-galactosida se alpha-2,6 sialyltransferase promoter,
insulin-like growth factor binding protein (IGFBP-1) promoter,
aldolase B promoter, human transferrin promoter, and collagen type
I promoter.
[0103] Exemplary tissue-specific expression elements for the
prostate include but are not limited to the prostatic acid
phosphatase (PAP) promoter, prostatic secretory protein of 94 (P SP
94) promoter, prostate specific antigen complex promoter, and human
glandular kallikrein gene promoter (hgt-1).
[0104] Exemplary tissue-specific expression elements for gastric
tissue include but are not limited to the human H+/K+-ATPase alpha
subunit promoter.
[0105] Exemplary tissue-specific expression elements for the
pancreas include but are not limited to pancreatitis associated
protein promoter (PAP), elastase 1 transcriptional enhancer,
pancreas specific amylase and elastase enhancer promoter, and
pancreatic cholesterol esterase gene promoter.
[0106] Exemplary tissue-specific expression elements for the
endometrium include, but are not limited to, the uteroglobin
promoter.
[0107] Exemplary tissue-specific expression elements for adrenal
cells include, but are not limited to, cholesterol side-chain
cleavage (SCC) promoter.
[0108] Exemplary tissue-specific expression elements for the
general nervous system include, but are not limited to, gamma-gamma
enolase (neuron-specific enolase, NSE) promoter.
[0109] Exemplary tissue-specific expression elements for the brain
include, but are not limited to, the neurofilament heavy chain
(NF--H) promoter.
[0110] Exemplary tissue-specific expression elements for
lymphocytes include, but are not limited to, the human
CGL-1/granzyme B promoter, the terminal deoxy transferase (TdT),
lambda 5, VpreB, and lck (lymphocyte specific tyrosine protein
kinase p561ck) promoter, the humans CD2 promoter and its 3'
transcriptional enhancer, and the human NK and T cell specific
activation (NKG5) promoter.
[0111] Exemplary tissue-specific expression elements for the colon
include, but are not limited to, pp 60c-src tyrosine kinase
promoter, organ-specific neoantigens (OSNs) promoter, and colon
specific antigen-P promoter.
[0112] Exemplary tissue-specific expression elements for breast
cells include, but are not limited to, the human alpha-lactalbumin
promoter.
[0113] Exemplary tissue-specific expression elements for the lung
include, but are not limited to, the cystic fibrosis transmembrane
conductance regulator (CFTR) gene promoter.
[0114] Other elements aiding specificity of expression in a tissue
of interest can include secretion leader sequences, enhancers,
nuclear localization signals, endosmolytic peptides, etc.
Preferably, these elements are derived from the tissue of interest
to aid specificity.
[0115] In general, the in vivo expression element shall include, as
necessary, 5' non-transcribing and 5' non-translating sequences
involved with the initiation of transcription. They optionally
include enhancer sequences or upstream activator sequences.
Methods and Materials for Production of miRNA
[0116] The miRNA can be isolated from cells or tissues,
recombinantly produced, or synthesized in vitro by a variety of
techniques well known to one of ordinary skill in the art.
[0117] In one embodiment, miRNA is isolated from cells or tissues.
Techniques for isolating miRNA from cells or tissues are well known
to one of ordinary skill in the art. For example, miRNA can be
isolated from total RNA using the mirVana miRNA isolation kit from
Ambion, Inc. Another techniques utilize[[s]] the flashPAGE.TM.
Fractionator System (Ambion, Inc.) for PAGE purification of small
nucleic acids.
[0118] The miRNA can be obtained by preparing a recombinant version
thereof (i.e., by using the techniques of genetic engineering to
produce a recombinant nucleic acid which can then be isolated or
purified by techniques well known to one of ordinary skill in the
art). This embodiment involves growing a culture of host cells in a
suitable culture medium, and purifying the miRNA from the cells or
the culture in which the cells are grown. For example, the methods
include a process for producing a miRNA in which a host cell
containing a suitable expression vector that includes a nucleic
acid encoding an miRNA is cultured under conditions that allow
expression of the encoded miRNA. In a preferred embodiment the
nucleic acid encodes let-7. The miRNA can be recovered from the
culture, from the culture medium or from a lysate prepared from the
host cells, and further purified. The host cell can be a higher
eukaryotic host cell such as a mammalian cell, a lower eukaryotic
host cell such as a yeast cell, or the host cell can be a
prokaryotic cell such as a bacterial cell. Introduction of a vector
containing the nucleic acid encoding the miRNA into the host cell
can be effected by calcium phosphate transfection, DEAE, dextran
mediated transfection, or electroporation (Davis, L. et al., Basic
Methods in Molecular Biology (1986)).
[0119] Any host/vector system can be used to express one or more of
the miRNAs. These include, but are not limited to, eukaryotic hosts
such as HeLa cells and yeast, as well as prokaryotic host such as
E. coli and B. subtilis. MiRNA can be expressed in mammalian cells,
yeast, bacteria, or other cells where the miRNA gene is under the
control of an appropriate promoter. Appropriate cloning and
expression vectors for use with prokaryotic and eukaryotic hosts
are described by Sambrook, et al., in Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989).
In the preferred embodiment, the miRNA is expressed in mammalian
cells. Examples of mammalian expression systems include C 127,
monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney
293 cells, human epidermal A431 cells, human Colo205 cells, 3T3
cells, CV-1 cells, other transformed primate cell lines, normal
diploid cells, cell strains derived from in vitro culture of
primary tissue, primary explants, HeLa cells, mouse L cells, BHK,
HL-60, U937, HaK or Jurkat cells. Mammalian expression vectors will
comprise an origin of replication, a suitable promoter,
polyadenylation site, transcriptional termination sequences, and 5'
flanking nontranscribed sequences. DNA sequences derived from the
SV40 viral genome, for example, SV40 origin, early promoter,
enhancer, splice, and polyadenylation sites may be used to provide
the required nontranscribed genetic elements. Potentially suitable
yeast strains include Saccharomyces cerevisiae, Schizosaccharomyces
pornbe, Kluyveromyces strains, Candida, or any yeast strain capable
of expressing miRNA. Potentially suitable bacterial strains include
Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any
bacterial strain capable of expressing miRNA.
[0120] In a preferred embodiment, genomic DNA encoding let-7 is
isolated, the genomic DNA is expressed in a mammalian expression
system, RNA is purified and modified as necessary for
administration to a patient. In a preferred embodiment the let-7 is
in the form of a pre-miRNA, which can be modified as desired (i.e.
for increased stability or cellular uptake).
[0121] Knowledge of DNA sequences of miRNA allows for modification
of cells to permit or increase expression of an endogenous miRNA.
Cells can be modified (e.g., by homologous recombination) to
provide increased miRNA expression by replacing, in whole or in
part, the naturally occurring promoter with all or part of a
heterologous promoter so that the cells express the miRNA at higher
levels. The heterologous promoter is inserted in such a manner that
it is operatively linked to the desired miRNA encoding sequences.
See, for example, PCT International Publication No. WO 94/12650 by
Transkaryotic Therapies, Inc., PCT International Publication No. WO
92/20808 by Cell Genesys, Inc., and PCT International Publication
No. WO 91/09955 by Applied Research Systems. Cells also may be
engineered to express an endogenous gene comprising the miRNA under
the control of inducible regulatory elements, in which case the
regulatory sequences of the endogenous gene may be replaced by
homologous recombination. Gene activation techniques are described
in U.S. Pat. No. 5,272,071 to Chappel; U.S. Pat. No. 5,578,461 to
Sherwin et al.; PCT/US92/09627 (WO93/09222) by Selden et al.; and
PCT/US90/06436 (WO91/06667) by Skoultchi et al.
[0122] The miRNA may be prepared by culturing transformed host
cells under culture conditions suitable to express the miRNA. The
resulting expressed miRNA may then be purified from such culture
(i.e., from culture medium or cell extracts) using known
purification processes, such as gel filtration and ion exchange
chromatography. The purification of the miRNA may also include an
affinity column containing agents which will bind to the protein;
one or more column steps over such affinity resins as concanavalin
A-agarose, heparin-Toyopearl.TM. or Cibacrom blue 3GA
Sepharose.TM.; one or more steps involving hydrophobic interaction
chromatography using such resins as phenyl ether, butyl ether, or
propyl ether; immunoaffinity chromatography, or complementary cDNA
affinity chromatography.
[0123] The miRNA may also be expressed as a product of transgenic
animals, which are characterized by somatic or germ cells
containing a nucleotide sequence encoding the miRNA. A vector
containing DNA encoding miRNA and appropriate regulatory elements
can be inserted in the germ line of animals using homologous
recombination (Capecchi, Science 244:1288-1292 (1989)), such that
the express the miRNA. Transgenic animals, preferably non-human
mammals, are produced using methods as described in U.S. Pat. No.
5,489,743 to Robinson, et al., and PCT Publication No. WO 94/28122
by Ontario Cancer Institute. miRNA can be isolated from cells or
tissue isolated from transgenic animals as discussed above.
[0124] In a preferred embodiment, the miRNA can be obtained
synthetically, for example, by chemically synthesizing a nucleic
acid by any method of synthesis known to the skilled artisan. The
synthesized miRNA can then be purified by any method known in the
art. Methods for chemical synthesis of nucleic acids include, but
are not limited to, in vitro chemical synthesis using
phosphotriester, phosphate or phosphoramidite cheminstry and solid
phase techniques, or via deosynucleoside H-phosphonate
intermediates (see U.S. Pat. No. 5,705,629 to Bhongle).
[0125] In some circumstances, for example, where increased nuclease
stability is desired, nucleic acids having nucleic acid analogs
and/or modified internucleoside linkages may be preferred. Nucleic
acids containing modified internucleoside linkages may also be
synthesized using reagents and methods that are well known in the
art. For example, methods of synthesizing nucleic acids containing
phosphonate phosphorothioate, phosphorodithioate, phosphoramidate
methoxyethyl phosphoramidate, formacetal, thioformacetal,
diisopropylsilyl, acetamidate, carbamate, dimethylene-sulfide
(--CH2-S--CH2), dimethylene-sulkodde (--CH2-SO--CH2),
dimethylene-sulfone (--CH2-SO2-CH2), 2'-0-alkyl, and
2'-deoxy-2'-fluoro phosphorothioate internucleoside linkages are
well known in the art (see Uhlmann et al., 1990, Chem. Rev.
90:543-584; Schneider et al., 1990, Tetrahedron Lett. 31:335 and
references cited therein). U.S. Pat. Nos. 5,614,617 and 5,223,618
to Cook, et al., 5,714,606 to Acevedo, et al., 5,378,825 to Cook,
et al., 5,672,697 and 5,466,786 to Buhr, et al., 5,777,092 to Cook,
et al., 5,602,240 to De Mesmaeker, et al., 5,610,289 to Cook, et
al. and 5,858,988 to Wang, also describe nucleic acid analogs for
enhanced nuclease stability and cellular uptake.
Formulations
[0126] The nucleic acids described above are preferably employed
for therapeutic uses in combination with a suitable pharmaceutical
carrier. Such compositions comprise an effective amount of the
compound, and a pharmaceutically acceptable carrier or excipient.
The formulation is made to suit the mode of administration.
Pharmaceutically acceptable carriers are determined in part by the
particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions containing the nucleic acids some of which are
described herein.
[0127] It is understood by one of ordinary skill in the art that
nucleic acids administered in vivo are taken up and distributed to
cells and tissues (Huang, et al., FEBS Lett. 558(1-3):69-73
(2004)). For example, Nyce et al. have shown that antisense
oligodeoxynucleotides (ODNs) when inhaled bind to endogenous
surfactant (a lipid produced by lung cells) and are taken up by
lung cells without a need for additional carrier lipids (Nyce and
Metzger, Nature, 385:721-725 (1997). Small nucleic acids are
readily taken up into T24 bladder carcinoma tissue culture cells
(Ma, et al., Antisense Nucleic Acid Drug Dev. 8:415-426 (1998).
siRNAs have been used for therapeutic silencing of an endogenous
genes by systemic administration (Soutschek, et al., Nature 432,
173-178 (2004)).
[0128] The nucleic acids described above may be in a formulation
for administration topically, locally or systemically in a suitable
pharmaceutical carrier. Remington's Pharmaceutical Sciences, 15th
Edition by E. W. Martin (Mark Publishing Company, 1975), discloses
typical carriers and methods of preparation. The nucleic acids may
also be encapsulated in suitable biocompatible microcapsules,
microparticles or microspheres formed of biodegradable or
non-biodegradable polymers or proteins or liposomes for targeting
to cells. Such systems are well known to those skilled in the art
and may be optimized for use with the appropriate nucleic acid.
[0129] Various methods for nucleic acid delivery are described, for
example in Sambrook et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York; and Ausubel et
al., 1994, Current Protocols in Molecular Biology, John Wiley &
Sons, New York. Such nucleic acid delivery systems comprise the
desired nucleic acid, by way of example and not by limitation, in
either "naked" form as a "naked" nucleic acid, or formulated in a
vehicle suitable for delivery, such as in a complex with a cationic
molecule or a liposome forming lipid, or as a component of a
vector, or a component of a pharmaceutical composition. The nucleic
acid delivery system can be provided to the cell either directly,
such as by contacting it with the cell, or indirectly, such as
through the action of any biological process. By way of example,
and not by limitation, the nucleic acid delivery system can be
provided to the cell by endocytosis, receptor targeting, coupling
with native or synthetic cell membrane fragments, physical means
such as electroporation, combining the nucleic acid delivery system
with a polymeric carrier such as a controlled release film or
nanoparticle or microparticle, using a vector, injecting the
nucleic acid delivery system into a tissue or fluid surrounding the
cell, simple diffusion of the nucleic acid delivery system across
the cell membrane, or by any active or passive transport mechanism
across the cell membrane. Additionally, the nucleic acid delivery
system can be provided to the cell using techniques such as
antibody-related targeting and antibody-mediated immobilization of
a viral vector.
[0130] Formulations for topical administration may include
ointments, lotions, creams, gels, drops, suppositories, sprays,
liquids and powders. Conventional pharmaceutical carriers, aqueous,
powder or oily bases, and thickeners can be used as desired.
[0131] Formulations suitable for parenteral administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions, solutions or emulsions that can include suspending
agents, solubilizers, thickening agents, dispersing agents,
stabilizers, and preservatives. Formulations for injection may be
presented in unit dosage form, e.g., in ampules or in multi-dose
containers, with an added preservative.
[0132] Preparations include sterile aqueous or nonaqueous
solutions, suspensions and emulsions, which can be isotonic with
the blood of the subject in certain embodiments. Examples of
nonaqueous solvents are polypropylene glycol, polyethylene glycol,
vegetable oil such as olive oil, sesame oil, coconut oil, arachis
oil, peanut oil, mineral oil, injectable organic esters such as
ethyl oleate, or fixed oils including synthetic mono or
di-glycerides. Aqueous carriers include water, alcoholic/aqueous
solutions, emulsions or suspensions, including saline and buffered
media. Parenteral vehicles include sodium chloride solution,
1,3-butandiol, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's or fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose).
[0133] Preservatives and other additives may also be present such
as, for example, antimicrobials, antioxidants, chelating agents and
inert gases. In addition, sterile, fixed oils are conventionally
employed as a solvent or suspending medium. For this purpose any
bland fixed oil may be employed including synthetic mono- or
di-glycerides. In addition, fatty acids such as oleic acid may be
used in the preparation of injectables. Carrier formulation can be
found in Remington's Pharmaceutical Sciences, Mack Publishing Co.,
Easton, Pa. Those of skill in the art can readily determine the
various parameters for preparing and formulating the compositions
without resort to undue experimentation.
[0134] The nucleic acids alone or in combination with other
suitable components, can also be made into aerosol formulations
(i.e., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, and
nitrogen. For administration by inhalation, the nucleic acids are
conveniently delivered in the form of an aerosol spray presentation
from pressurized packs or a nebulizer, with the use of a suitable
propellant.
[0135] In some embodiments, the nucleic acids described above may
include pharmaceutically acceptable carriers with formulation
ingredients such as salts, carriers, buffering agents, emulsifiers,
diluents, excipients, chelating agents, fillers, drying agents,
antioxidants, antimicrobials, preservatives, binding agents,
bulking agents, silicas, solubilizers, or stabilizers. In one
embodiment, the nucleic acids are conjugated to lipophilic groups
like cholesterol and lauric and lithocholic acid derivatives with
C32 functionality to improve cellular uptake. For example,
cholesterol has been demonstrated to enhance uptake and serum
stability of siRNA in vitro (Lorenz, et al., Bioorg. Med. Chem.
Lett. 14(19):4975-4977 (2004)) and in vivo (Soutschek, et al.,
Nature 432(7014):173178 (2004)). In addition, it has been shown
that binding of steroid conjugated oligonucleotides to different
lipoproteins in the bloodstream, such as LDL, protect integrity and
facilitate biodistribution (Rump, et al., Biochem. Pharmacol.
59(11):1407-1416 (2000)). Other groups that can be attached or
conjugated to the nucleic acids described above to increase
cellular uptake, include, but are not limited to,
acridinederivatives; cross-linkers such as psoralen derivatives,
azidophenacyl, proflavin, and azidoproflavin; artificial
endonucleases; metal complexes such as EDTA-Fe(II) and
porphyrin-Fe(II); alkylating moieties,; nucleases such as alkaline
phosphatase; terminal transferases; abzymes; cholesteryl moieties;
lipophilic carriers; peptide conjugates; long chain alcohols;
phosphate esters; radioactive markers; non-radioactive markers;
carbohydrates; and polylysine or other polyamines. U.S. Pat. No.
6,919,208 to Levy, et al., also described methods for enhanced
delivery of nucleic acids molecules.
[0136] These pharmaceutical formulations may be manufactured in a
manner that is itself known, e.g., by means of conventional mixing,
dissolving, granulating, levigating, emulsifying, encapsulating,
entrapping or lyophilizing processes.
[0137] The formulations described herein of the nucleic acids
embrace fusions of the nucleic acids or modifications of the
nucleic acids, wherein the nucleic acid is fused to another moiety
or moieties, e.g., targeting moiety or another therapeutic agent.
Such analogs may exhibit improved properties such as activity
and/or stability. Examples of moieties which may be linked or
unlinked to the nucleic acid include, for example, targeting
moieties which provide for the delivery of nucleic acid to specific
cells, e.g., antibodies to pancreatic cells, immune cells, lung
cells or any other preferred cell type, as well as receptor and
ligands expressed on the preferred cell type. Preferably, the
moieties target cancer or tumor cells. For example, since cancer
cells have increased consumption of glucose, the nucleic acids can
be linked to glucose molecules. Monoclonal humanized antibodies
that target cancer or tumor cells are preferred moieties and can be
linked or unlinked to the nucleic acids. In the case of cancer
therapeutics, the target antigen is typically a protein that is
unique and/or essential to the tumor cells (e.g., the receptor
protein HER-2).
II. Methods of Treatment
[0138] The compositions are administered to a patient in need of
treatment of at least one symptom or manifestation (since disease
can occur/progress in the absence of symptoms) of cancer where it
acts as a radiation or chemotherapy sensitizer. The compositions
described herein can be administered to a subject prior to
administration of a cytotoxic therapy in an amount effective to
sensitize cells or tissues to be treated to the effects of the
cytotoxic therapy. In one embodiment the cytotoxic therapy is
radiotherapy. In another embodiment the cytotoxic therapy is
chemotherapy. Sensitization describes a condition of the cells or
tissues to be treated in which prior administration of the
compositions described herein increases at least one effect of the
cytotoxic therpapy on the cells or tissues relative to cells or
tissues not receiving prior administration of the compositions
described herein. The increased effect may be on reduction of tumor
size, reduction in cell proliferation of a tumor, inhibition of
angiogenesis, inhibition of metastasis, or improvement of at least
one symptom or manifestation of the disease.
[0139] Aberrant expression of oncogenes is a hallmark of cancer
such as lung cancer. In another embodiment, the compositions are
administered in an effective amount to inhibit gene expression of
one or more oncogenes. In yet another embodiment, the compositions
are administered in an effective amount to inhibit gene expression
of RAS, MYC, and/or BCL-2.
[0140] Methods for treatment or prevention of at least one symptom
or manifestation of cancer are also described including
administration of an effective amount of a composition containing a
nucleic acid molecule to alleviate at least one symptom or decrease
at least one manifestation. In a preferred embodiment, the cancer
is lung cancer. The compositions described herein can be
administered in effective dosages alone or in combination with
adjuvant cancer therapy such as surgery, chemotherapy,
radiotherapy, thermotherapy, immunotherapy, hormone therapy and
laser therapy, to provide a beneficial effect, e.g. reduce tumor
size, reduce cell proliferation of the tumor, inhibit angiogenesis,
inhibit metastasis, or otherwise improve at least one symptom or
manifestation of the disease.
Method of Administration
[0141] In general, methods of administering nucleic acids are well
known in the art. In particular, the routes of administration
already in use for nucleic acid therapeutics, along with
formulations in current use, provide preferred routes of
administration and formulation for the nucleic acids described
above.
[0142] Nucleic acid compositions can be administered by a number of
routes including, but not limited to: oral, intravenous,
intraperitoneal, intramuscular, transdermal, subcutaneous, topical,
sublingual, or rectal means. Nucleic acids can also be administered
via liposomes. Such administration routes and appropriate
formulations are generally known to those of skill in the art.
[0143] Administration of the formulations described herein may be
accomplished by any acceptable method which allows the miRNA or
nucleic acid encoding the miRNA to reach its target. The particular
mode selected will depend of course, upon factors such as the
particular formulation, the severity of the state of the subject
being treated, and the dosage required for therapeutic efficacy. As
generally used herein, an "effective amount" of a nucleic acid is
that amount which is able to treat one or more symptoms of cancer
or related disease, reverse the progression of one or more symptoms
of cancer or related disease, halt the progression of one or more
symptoms of cancer or related disease, or prevent the occurrence of
one or more symptoms of cancer or related disease in a subject to
whom the formulation is administered, as compared to a matched
subject not receiving the compound or therapeutic agent. The actual
effective amounts of drug can vary according to the specific drug
or combination thereof being utilized, the particular composition
formulated, the mode of administration, and the age, weight,
condition of the patient, and severity of the symptoms or condition
being treated.
[0144] Any acceptable method known to one of ordinary skill in the
art may be used to administer a formulation to the subject. The
administration may be localized (i.e., to a particular region,
physiological system, tissue, organ, or cell type) or systemic,
depending on the condition being treated.
[0145] Injections can be e.g., intravenous, intradermal,
subcutaneous, intramuscular, or intraperitoneal. The composition
can be injected intradermally for treatment or prevention of
cancer, for example. In some embodiments, the injections can be
given at multiple locations. Implantation includes inserting
implantable drug delivery systems, e.g., microspheres, hydrogels,
polymeric reservoirs, cholesterol matrixes, polymeric systems,
e.g., matrix erosion and/or diffusion systems and non-polymeric
systems, e.g., compressed, fused, or partially-fused pellets.
Inhalation includes administering the composition with an aerosol
in an inhaler, either alone or attached to a carrier that can be
absorbed. For systemic administration, it may be preferred that the
composition is encapsulated in liposomes.
[0146] Preferably, the agent and/or nucleic acid delivery system
are provided in a manner which enables tissue-specific uptake of
the agent and/or nucleic acid delivery system. Techniques include
using tissue or organ localizing devices, such as wound dressings
or transdermal delivery systems, using invasive devices such as
vascular or urinary catheters, and using interventional devices
such as stents having drug delivery capability and configured as
expansive devices or stent grafts.
[0147] The formulations may be delivered using a bioerodible
implant by way of diffusion or by degradation of the polymeric
matrix. In certain embodiments, the administration of the
formulation may be designed so as to result in sequential exposures
to the miRNA over a certain time period, for example, hours, days,
weeks, months or years. This may be accomplished, for example, by
repeated administrations of a formulation or by a sustained or
controlled release delivery system in which the miRNA is delivered
over a prolonged period without repeated administrations.
Administration of the formulations using such a delivery system may
be, for example, by oral dosage forms, bolus injections,
transdermal patches or subcutaneous implants. Maintaining a
substantially constant concentration of the composition may be
preferred in some cases.
[0148] Other delivery systems suitable include, but are not limited
to, time-release, delayed release, sustained release, or controlled
release delivery systems. Such systems may avoid repeated
administrations in many cases, increasing convenience to the
subject and the physician. Many types of release delivery systems
are available and known to those of ordinary skill in the art. They
include, for example, polymer-based systems such as polylactic
and/or polyglycolic acids, polyanhydrides, polycaprolactones,
copolyoxalates, polyesteramides, polyorthoesters,
polyhydroxybutyric acid, and/or combinations of these.
Microcapsules of the foregoing polymers containing nucleic acids
are described in, for example, U.S. Pat. No. 5,075,109. Other
examples include nonpolymer systems that are lipid-based including
sterols such as cholesterol, cholesterol esters, and fatty acids or
neutral fats such as mono-, di- and triglycerides; hydrogel release
systems; liposome-based systems; phospholipid based-systems;
silastic systems; peptide based systems; wax coatings; compressed
tablets using conventional binders and excipients; or partially
fused implants. Specific examples include, but are not limited to,
erosional systems in which the miRNA is contained in a formulation
within a matrix (for example, as described in U.S. Pat. Nos.
4,452,775, 4,675,189, 5,736,152, 4,667,013, 4,748,034 and
5,239,660), or diffusional systems in which an active component
controls the release rate (for example, as described in U.S. Pat.
Nos. 3,832,253, 3,854,480, 5,133,974 and 5,407,686). The
formulation may be as, for example, microspheres, hydrogels,
polymeric reservoirs, cholesterol matrices, or polymeric systems.
In some embodiments, the system may allow sustained or controlled
release of the composition to occur, for example, through control
of the diffusion or erosion/degradation rate of the formulation
containing the miRNA. In addition, a pump-based hardware delivery
system may be used to deliver one or more embodiments.
[0149] Examples of systems in which release occurs in bursts
includes, e.g., systems in which the composition is entrapped in
liposomes which are encapsulated in a polymer matrix, the liposomes
being sensitive to specific stimuli, e.g., temperature, pH, light
or a degrading enzyme and systems in which the composition is
encapsulated by an ionically-coated microcapsule with a
microcapsule core degrading enzyme. Examples of systems in which
release of the inhibitor is gradual and continuous include, e.g.,
erosional systems in which the composition is contained in a form
within a matrix and effusional systems in which the composition
permeates at a controlled rate, e.g., through a polymer. Such
sustained release systems can be e.g., in the form of pellets, or
capsules.
[0150] Use of a long-term release implant may be particularly
suitable in some embodiments. "Long-term release," as used herein,
means that the implant containing the composition is constructed
and arranged to deliver therapeutically effective levels of the
composition for at least 30 or 45 days, and preferably at least 60
or 90 days, or even longer in some cases. Long-term release
implants are well known to those of ordinary skill in the art, and
include some of the release systems described above.
[0151] Dosages for a particular patient can be determined by one of
ordinary skill in the art using conventional considerations, (e.g.
by means of an appropriate, conventional pharmacological protocol).
A physician may, for example, prescribe a relatively low dose at
first, subsequently increasing the dose until an appropriate
response is obtained. The dose administered to a patient is
sufficient to effect a beneficial therapeutic response in the
patient over time, or, e.g., to reduce symptoms, or other
appropriate activity, depending on the application. The dose is
determined by the efficacy of the particular formulation, and the
activity, stability or serum half-life of the miRNA employed and
the condition of the patient, as well as the body weight or surface
area of the patient to be treated. The size of the dose is also
determined by the existence, nature, and extent of any adverse
side-effects that accompany the administration of a particular
vector, and formulation, in a particular patient.
[0152] Therapeutic compositions comprising one or more nucleic
acids are optionally tested in one or more appropriate in vitro
and/or in vivo animal models of disease, to confirm efficacy,
tissue metabolism, and to estimate dosages, according to methods
well known in the art. In particular, dosages can be initially
determined by activity, stability or other suitable measures of
treatment versus non-treatment (e.g., comparison of treated vs.
untreated cells or animal models), in a relevant assay.
Formulations are administered at a rate determined by the LD50 of
the relevant formulation, and/or observation of any side-effects of
the nucleic acids at various concentrations, e.g., as applied to
the mass and overall health of the patient. Administration can be
accomplished via single or divided doses.
[0153] In vitro models can be used to determine the effective doses
of the nucleic acids as a potential cancer treatment. Suitable in
vitro models include, but are not limited to, proliferation assays
of cultured tumor cells, growth of cultured tumor cells in soft
agar (see Freshney, (1987) Culture of Animal Cells: A Manual of
Basic Technique, Wily-Liss, New York, N.Y. Ch 18 and Ch 21), tumor
systems in nude mice as described in Giovanella et al., J. Natl.
Can. Inst., 52: 921-30 (1974), mobility and invasive potential of
tumor cells in Boyden Chamber assays as described in Pilkington et
al., Anticancer Res., 17: 4107-9 (1997), and angiogenesis assays
such as induction of vascularization of the chick chorioallantoic
membrane or induction of vascular endothelial cell migration as
described in Ribatta et al., Intl. J. Dev. Biol., 40: 1189-97
(1999) and Li et al., Clin. Exp. Metastasis, 17:423-9 (1999),
respectively. Suitable tumor cells lines are available, e.g. from
American Type Tissue Culture Collection catalogs.
[0154] In vivo models are the preferred models to determine the
effective doses of nucleic acids described above as potential
cancer treatments. Suitable in vivo models include, but are not
limited to, mice that carry a mutation in the KRAS oncogene
(Lox-Stop-Lox K-RasG12D mutants, Kras2tm4TH) available from the
National Cancer Institute (NCI) Frederick Mouse Repository. Other
mouse models known in the art and that are available include but
are not limited to models for gastrointestinal cancer,
hematopoietic cancer, lung cancer, mammary gland cancer, nervous
system cancer, ovarian cancer, prostate cancer, skin cancer,
cervical cancer, oral cancer, and sarcoma cancer (see
http://emice.nci.nih.gov/mouse_models/).
[0155] In determining the effective amount of the miRNA to be
administered in the treatment or prophylaxis of disease the
physician evaluates circulating plasma levels, formulation
toxicities, and progression of the disease.
[0156] The dose administered to a 70 kilogram patient is typically
in the range equivalent to dosages of currently-used therapeutic
antisense oligonucleotides such as Vitravene.RTM. (fomivirsen
sodium injection) which is approved by the FDA for treatment of
cytomegaloviral RNA, adjusted for the altered activity or serum
half-life of the relevant composition.
[0157] The formulations described herein can supplement treatment
conditions by any known conventional therapy, including, but not
limited to, antibody administration, vaccine administration,
administration of cytotoxic agents, natural amino acid
polypeptides, nucleic acids, nucleotide analogues, and biologic
response modifiers. Two or more combined compounds may be used
together or sequentially. For example, the nucleic acids can also
be administered in therapeutically effective amounts as a portion
of an anti-cancer cocktail. An anti-cancer cocktail is a mixture of
the oligonucleotide or modulator with one or more anti-cancer drugs
in addition to a pharmaceutically acceptable carrier for delivery.
The use of anti-cancer cocktails as a cancer treatment is routine.
Anti-cancer drugs that are well known in the art and can be used as
a treatment in combination with the nucleic acids described herein
include, but are not limited to: Actinomycin D, Aminoglutethimide,
Asparaginase, Bleomycin, Busulfan, Carboplatin, Carmustine,
Chlorarnbucil, Cisplatin (cis-DDP), Cyclophosphamide, Cytarabine
HCl (Cytosine arabinoside), Dacarbazine, Dactinomycin, Daunorubicin
HCl, Doxorubicin HCl, Estramustine phosphate sodium, Etoposide
(V16-213), Floxuridine, 5-Fluorouracil (5-Fu), Flutamide,
Hydroxyurea (hydroxycarbamide), Ifosfamide, Interferon Alpha-2a,
Interferon Alpha-2b, Leuprolide acetate (LHRH-releasing factor
analog), Lomustine, Mechlorethamine HCl (nitrogen mustard),
Meiphalan, Mercaptopurine, Mesna, Methotrexate (MTX), Mitomycin,
Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,
Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastine
sulfate, Vincristine sulfate, Amsacrine, Azacitidine,
Hexamethylmelamine, Interleukin-2, Mitoguazone, Pentostatin,
Semustine, Tenipo side, and Vindesine sulfate.
Diseases to be Treated
[0158] Cancer treatments promote tumor regression by inhibiting
tumor cell proliferation, inhibiting angiogenesis (growth of new
blood vessels that is necessary to support tumor growth) and/or
prohibiting metastasis by reducing tumor cell motility or
invasiveness. Therapeutic formulations described herein may be
effective in adult and pediatric oncology including in solid phase
tumors/malignancies, locally advanced tumors, human soft tissue
sarcomas, metastatic cancer, including lymphatic metastases, blood
cell malignancies including multiple myeloma, acute and chronic
leukemias, and lymphomas, head and neck cancers including mouth
cancer, larynx cancer and thyroid cancer, lung cancers including
small cell carcinoma and non-small cell cancers, breast cancers
including small cell carcinoma and ductal carcinoma,
gastrointestinal cancers including esophageal cancer, stomach
cancer, colon cancer, colorectal cancer and polyps associated with
colorectal neoplasia, pancreatic cancers, liver cancer, urologic
cancers including bladder cancer and prostate cancer, malignancies
of the female genital tract including ovarian carcinoma, uterine
(including endometrial) cancers, and solid tumor in the ovarian
follicle, kidney cancers including renal cell carcinoma, brain
cancers including intrinsic brain tumors, neuroblastoma, astrocytic
brain tumors, gliomas, metastatic tumor cell invasion in the
central nervous system, bone cancers including osteomas, skin
cancers including malignant melanoma, tumor progression of human
skin keratinocytes, squamous cell carcinoma, basal cell carcinoma,
hemangiopericytoma and Karposits sarcoma. Therapeutic formulations
can be administered in therapeutically effective dosages alone or
in combination with adjuvant cancer therapy such as surgery,
chemotherapy, radiotherapy, thermotherapy, immunotherapy, hormone
therapy and laser therapy, to provide a beneficial effect, e.g.
reducing tumor size, slowing rate of tumor growth, reducing cell
proliferation of the tumor, promoting cancer cell death, inhibiting
angiongenesis, inhibiting metastasis, or otherwise improving
overall clinical condition, without necessarily eradicating the
cancer.
[0159] Cancers include, but are not limited to, biliary tract
cancer; bladder cancer; breast cancer; brain cancer including
glioblastomas and medulloblastomas; cervical cancer;
choriocarcinoma; colon cancer including colorectal carcinomas;
endometrial cancer; esophageal cancer; gastric cancer; head and
neck cancer; hematological neoplasms including acute lymphocytic
and myelogenous leukemia, multiple myeloma, AIDS-associated
leukemias and adult T-cell leukemia lymphoma; intraepithelial
neoplasms including Bowen's disease and Pagers disease; liver
cancer; lung cancer including small cell lung cancer and non-small
cell lung cancer; lymphomas including Hodgkin's disease and
lymphocytic lymphomas; neuroblastomas; oral cancer including
squamous cell carcinoma; osteosarcomas; ovarian cancer including
those arising from epithelial cells, stromal cells, germ cells and
mesenchymal cells; pancreatic cancer; prostate cancer; rectal
cancer; sarcomas including leiomyosarcoma, rhabdomyosarcoma,
liposarcoma, fibrosarcoma, synovial sarcoma and osteosarcoma; skin
cancer including melanomas, Kaposi's sarcoma, basocellular cancer,
and squamous cell cancer; testicular cancer including germinal
tumors such as seminoma, non-seminoma (teratomas,
choriocarcinomas), stromal tumors, and germ cell tumors; thyroid
cancer including thyroid adenocarcinoma and medullar carcinoma;
transitional cancer and renal cancer including adenocarcinoma and
Wilms tumor. In a preferred embodiment, the formulations are
administered for treatment or prevention of lung cancer.
[0160] In addition, therapeutic nucleic acids may be used for
prophylactic treatment of cancer. There are hereditary conditions
and/or environmental situations (e.g. exposure to carcinogens)
known in the art that predispose an individual to developing
cancers. Under these circumstances, it may be beneficial to treat
these individuals with therapeutically effective doses of the
nucleic acids to reduce the risk of developing cancers. In one
embodiment, a nucleic acid in a suitable formulation may be
administered to a subject who has a family history of cancer, or to
a subject who has a genetic predisposition for cancer. In other
embodiments, the nucleic acid in a suitable formulation is
administered to a subject who has reached a particular age, or to a
subject more likely to get cancer. In yet other embodiments, the
nucleic acid in a suitable formulation is administered to subjects
who exhibit symptoms of cancer (e.g., early or advanced). In still
other embodiments, the nucleic acid in a suitable formulation may
be administered to a subject as a preventive measure. In some
embodiments, the nucleic acid in a suitable formulation may be
administered to a subject based on demographics or epidemiological
studies, or to a subject in a particular field or career.
EXAMPLES
[0161] The present invention will be further understood by
reference to the following non-limiting examples.
Example 1
RAS is Regulated by the let-7 microRNA Family
[0162] C. elegans let-7, mir-48, mir-84 and mir-241 encode four
developmentally regulated miRNAs that comprise the let-7 family
(Lau et al., (2001) Science 294, 858-862; Lim et al., (2003) Genes
Dev 17, 991-1008; Reinhart et al., (2000) Nature 403, 901-906).
This family displays high sequence identity, with particular
conservation at the 5' end of the mature miRNAs (FIG. 10A, 10B).
The C. elegans let-7 family miRNAs, mir-84, plays a role in vulval
development, a model for dissecting RAS/MAP Kinase signaling (Wang
and Sternberg, (2001) Curr Top Dev Biol 51, 189-220). C. elegans
let-60/RAS is regulated by members of the let-7 family. let-7 and
mir-84 are complementary to multiple sites in the 3' UTR of
let-60/RAS. let-7 and mir-84 are expressed in a reciprocal manner
to let-60/RAS in the hypodermis and the vulva respectively.
[0163] let-7 and mir-84 genetically interact with let-60/RAS,
consistent with negative regulation of RAS expression by let-7 and
mir-84. The results demonstrate for the first time that miRNAs can
regulate RAS, a critical human oncogene. Moreover, all three human
RAS genes have let-7 complementary sites in their 3'UTRs that
subject the oncogenes to let-7 miRNA-mediated regulation in cell
culture. Lung tumor tissues display significantly reduced levels of
let-7 and significantly increased levels of RAS protein relative to
normal lung tissue, suggesting let-7 regulation of RAS as a
mechanism for let-7 in lung oncogenesis.
Experimental Procedures
[0164] Plasmid Constructs. PSJ840 (mir-84::gM was made by
amplifying 2.2 kb of genomic sequence (base pairs -2201 to -9)
upstream of the mature mir-84 sequence from N2 genomic DNA and
adding a Smal site and an Agel site to the 5' and 3' ends,
respectively, using the polymerase chain reaction (PCR) with
primers MIR84UP and MIR84DN (all primer sequences available upon
request). This product was digested with Smal and AgeI and then
cloned into the pPD95.70 vector digested with Smal and AgeI. The
mir-84 upstream DNA contained various sequence elements that are
also found in the let-7 upstream DNA. PSJo84 (mir-84(+++)) was made
by amplifying 3.0 kb of genomic sequence (base pairs -2201 to +792)
upstream and downstream of the mature mir-84 sequence including the
mir-84 sequence itself from N2 genomic DNA and adding Smal sites to
both the 5' and 3' ends using PCR with primers MIR84UP and
MIR84DN2. This product was ligated into the pCR4-TOPO vector using
the TOPO TA Cloning Kit as described by the manufacturer
(Invitrogen). The empty TOPO control vector was made by digesting
PSJo84 with EcoRI, extracting the 4 kb vector band and self
ligating it. The miR-84 deletion plasmid, 084.DELTA.84
(.DELTA.mir-84(+++)), was made by overlap extension PCR, starting
with two separate PCR reactions using primers MIR84UP with DEL84DN
and MIR84DN2 with DEL84UP and PSJo84 plasmid as template.
[0165] The two PCR products were purified (QIAGEN) and then used
together as template for the final PCR using primers MIR84UP and
MIR84DN2. This final product, identical to PSJo84 except for the
deletion of the 75 nt pre-miR84 encoding sequence, was ligated into
the pCR2.1-TOPO vector using the TOPO TA Cloning Kit (Invitrogen)
and called 084.DELTA.84. GFP60 was made by amplifying the 819 by of
genomic sequence encoding the let-60 3'UTR from N2 genomic DNA and
adding EcoRI and Spel sites to the 5' and 3' ends, respectively,
using PCR with primers 3LET60UP and 3LET60DN. This product was
digested with EcoRI and Spel and then cloned into the pPD95.70
vector (Fire Lab) digested with EcoRI and Spel, thus replacing the
unc-54 3'UTR found in pPD95.70 with the let-60 3'UTR. The green
fluorescent protein gene (GFP) followed by the let-60 3'UTR was
then amplified out of this plasmid and BglII and NotI sites were
added to the 5' and 3' ends respectively using PCR with primers
BGLGFP and 3UTRNOT. This PCR product was digested with BglII and
NotI and ligated into PB255 digested with BglII and NotI, resulting
in the plasmid GFP60 containing the lin-31 promoter and enhancer
driving GFP with the let-60 3'UTR. GFP54 was made by amplifying gfp
followed by the unc-54 3'UTR out of pPD95.70 using PCR with primers
BGLGFP and 3UTRNOT. This PCR product was digested and ligated into
PB255 digested with BglII and Nod, resulting in the plasmid GFP54
containing the lin-31 promoter and enhancer driving GFP with the
unc-54 3'UTR. pGL3-NRAS S and pGL3-NRAS L were made by amplifying
the entire 1140 by shorter form of the H.s. NRAS 3'UTR out of a
IMAGE cDNA clone (accession # AI625442), or the entire 3616 by
longer form of the Hs. NRAS 3'UTR (excluding the first 43 bp) from
H.s. genomic DNA, and adding NheI sites to the ends by PCR using
primers SMJ100 and SMJ101 or SMJ102 and SMJ103 respectively. These
products were digested with NheI and ligated into pGL3-Control
(Promega) digested with XbaI and treated with CIP, resulting in
Rrluc-expressing plasmids containing either the short or long form
of the H.s. NRAS 3'UTR. To generate pFS1047, containing the
col-10-lacZ-let-60 3'UTR reporter gene, the entire let-60 3'UTR was
subcloned into the Sacll and NcoI sites of plasmid B29 (Wightman,
B., Ha, I., and Ruvkun, G. (1993) Cell 75, 855-862).
[0166] let-60; let-7 Double Mutants. let-60(n2021); let-7(mn112)
double mutant animals were generated by crossing let-60(n2021)
heterozygotes with let-7(mn112) unc-3/0; mnDp1 hemiyzgotes. From
this cross, 180 individual homozygous let-7(mn112) F2 animals were
female, as determined by their Unc-phenotype, due to the tight
linkage of the let-7(mn112) to unc-3. Of these, 10 survived into
adulthood and produced eggs. The resulting progeny from these
animals died as larvae with the rod-like phenotype that is
characteristic of let-60 mutant animals, thus showing that these
animals all contained the let-60(n2021) mutation, and that adult
survival was likely due to the let-60(n2021) mutation, confirming
our RNAi data. From 180 F2s, 25% (or about 45 animals) would have
been predicted to be let-60(n2021) homozygous. Since one saw
survival of only 10 of these animals, this indicates a suppression
of about 22%, similar to what was observed with RNAi. However,
because the brood sizes were low (usually about 10 hatched larvae
and several unhatched eggs); and because of a combination of larval
lethality as well as limited parental survival, double mutant lines
could not be established for further analysis; all progeny died as
larvae.
[0167] C. elegans and Transgenic reporter analysis. All animal
experiments were performed at room temperature or 20.degree. C.
unless stated otherwise. All experimental plasmids were injected in
animals at 50-100 ng/.mu.l. Two different markers, rol-6 (100
ng/.mu.l) and myo-3::gfp, (50 ng/.mu.l), were separately
co-injected with PSJo84: myo-3::gfp (50 ng/.mu.l) was co-injected
with 084.DELTA.84; and myo-2::gfp, (5 ng/.mu.l) was co-injected
with GFP60 and GFP54. These animals are mosaic for the transgenes.
To compare expression between individual lines, the percent
expression of GFP in each of the Pn.p cells was normalized relative
to the expression of the highest expressing Pn.p cell and
represented as a fraction of the highest expresser for each
individual line of animals. For each construct the average of the
lines was calculated along with the standard deviation for each
construct represented as error bars (mir-84::gfp n=239, gfp60 n=42
and gfp54 n=40). For the mir-84(+++) analysis, animals were
examined using DIC optics to score seam cell and vulval anatomy.
LacZ reporter analysis was as described (Vella et al., (2004) Genes
Dev 18, 132-137). The lin-41 3'UTR missing its LCSs (pFS1031) was
used as a control (Reinhart et al., (2000)). RNAi methods were
standard feeding procedures using synchronized Lls (Timmons et al.,
(2001) Gene 263, 103-112). All RNAi experiments were done in
parallel to an empty vector (L4440) feeding control. See
Supplemental Experimental procedures for details on the let-60;
let-7 double mutant cross.
[0168] let-7/RAS association in mammalian cells. HeLa S3 cells
grown in D-MEM (GIBCO) supplemented with 10% fetal bovine serum
(GIBCO) were cotransfected in 12-well plates using Lipofectamine
2000 (Invitrogen) according to the manufacturer's protocol using
1.0 .mu.g/well of Pp-luc-expressing plasmid (pGL3-Control from
Promega, pGL3-NRAS S pGL3-NRAS L and pGL3-KRAS) and 0.1 .mu.g/well
of Rr-luc-expressing plasmid (pRL-TK from Promega). 24 hrs post
transfection, the cells were harvested and assayed using the Dual
Luciferase assay as described by the manufacturer (Promega). HeLa
cells grown as above were transfected in 24-well plates with 30
pmoles of Anti-miR let-7 or negative control #1 inhibitors (Ambion)
using Lipofectamine 2000. Three days post-transfection, RAS
expression was monitored by immunofluorescence using an FITC
conjugated primary antibody against RAS protein (US Biological).
The resulting fluorescent signal was analyzed using the appropriate
filter set and was quantified using MetaMorph software. The
fluorescence intensity of 150-300 cells was typically measured in
one or a few viewing areas. The experiments with both the
precursors and the inhibitors were performed three times. The
photos represent single viewing fields from one of the experiments
and are representative of the triplicate experiment. Identically
grown HeLa cells were cotransfected in 24-well plates using
Lipofectamine 2000 (Invitrogen) according to the manufacturer's
protocol using 200 ng/well of Pp-luc-expressing plasmid
(pGL3-Control from Promega, pGL3-NRAS S pGL3-NRAS L and pGL3-KRAS).
48 hrs post transfection, the cells were harvested and assayed
using the Luciferase assay as described by the manufacturer
(Promega).
[0169] HepG2 cells grown in D-MEM (GIBCO) supplemented with 10%
fetal bovine serum (GIBCO) were transfected with 15 or 5 pmole of
Pre-miR Let-7c or negative control #1 Precursor miRNAs (Ambion) in
24-well plates using siPort Neo-FX (Ambion) according to the
manufacturer's protocol. Three days post-transfection, RAS
expression was monitored by immunofluorescence as described
above.
[0170] mRNA microarray analysis procedures used by Ambion, Inc.
Total RNA from tumor and normal adjacent tissue (NAT) samples from
3 breast cancer, 6 colon cancer, and 12 lung cancer patients was
isolated using the mirVana RNA Isolation Kit (Ambion). Twenty .mu.g
of each total RNA sample was fractionated by polyacrylamide gel
electrophoresis (PAGE) using a 15% denaturing polyacrylamide gel
and the miRNA fractions for each sample were recovered. The miRNAs
from all of the samples were subjected to a poly(A) polymerase
reaction wherein amine modified uridines were incorporated as part
of .about.40 nt long tails (Ambion). The tailed tumor samples were
fluorescently labeled using an amine-reactive Cy3 (Amersham) and
the normal adjacent tissue samples were labeled with Cy5
(Amersham). The fluorescently labeled miRNAs were purified by
glass-fiber filter binding and elution (Ambion) and the tumor and
normal adjacent tissue samples from the same patient were mixed.
Each sample mixture was hybridized for 14 hours with slides upon
which 167 miRNA probes were arrayed. The microarrays were washed
3.times.2 minutes (min) in 2.times.SSC and scanned using a GenePix
4000B (Axon). Fluorescence intensities for the Cy3- and Cy5-labeled
samples for each element were normalized by total Cy3 and Cy5
signal on the arrays. The normalized signal intensity for each
element was compared between the tumor and NAT samples from each
pair of patient samples and expressed as a log ratio of the tumor
to normal adjacent sample.
[0171] Northern Analysis. mir-84 northerns were performed as
described (Johnson et al., (2003) Dev Biol 259, 364-379). For human
tissues, 1 .mu.g of total RNA from the tumor and normal adjacent
tissues of patients 1 and 5 were fractionated by PAGE using a 15%
denaturing polyacrylamide gel. The RNA was transferred to a
positively charged nylon membrane by electroblotting at 200 mA in
0.5.times.TBE for 2 hours. The Northern blot was dried and then
incubated overnight in 10 ml of ULTRAhyb-Oligo (Ambion) with
10.sup.7 cpm of a radio-labeled transcript complementary to let-7c.
The blot was washed 3.times.10 min at room temperature in
2.times.SSC, 0.5% SDS and then 1.times.15 min at 42.degree. C. in
2.times.SSC, 0.5% SDS. Overnight phosphorimaging using the Storm
system (Amersham), revealed let-7c. The process was repeated using
a radio-labeled probe for 5S rRNA.
[0172] Lung tumor protein/northern/mRNA analysis. Total RNA and
protein were isolated from tumor and normal adjacent tissue samples
from three lung cancer patients using the mirVana PARIS Kit
(Ambion). let-7 miRNA and U6 snRNA were measured using the Northern
procedure described above. NRAS and B-actin mRNA as well as 18S
rRNA were quantified by real-time RT-PCR using primers specific to
each of the target RNAs. RAS and GAPDH protein were measured by
Western analysis using the RAS antibody described above and an
antibody for GAPDH (Ambion).
[0173] Accession numbers. The following sequences were searched for
3'UTR LCSs: Hs KRAS (Genbank M54968), Hs HRAS (NM176795), Hs NRAS
(BC005219). NRAS is known to exist as a 2 Kb and a 4.3 Kb form.
BC005219 represents the short form with a 1151 nt polyadenylated
3'UTR. Two human EST clones (Genbank BU177671 and BG388501) were
sequenced to obtain additional NRAS 3'UTR sequence. This revealed
that the NRAS 3'UTR exists in a 3642 nt polyadenylated 3'UTR
version, utilizing an alternative polyadenylation and cleavage
site, 2491 nt downstream of the first. This presumably corresponds
to the long NRAS form. The sequence was deposited with accession
numbers AY941100 and AY941101.
Results
[0174] Additional targets including let-60/RAS of the let-7 miRNA
in C. elegans. The let-7 miRNA is temporally expressed in C.
elegans (Johnson et al., (2003) Nature 426, 845-849; Pasquinelli et
al., (2000) Nature 408, 86-89; Reinhart et al., (2000) Nature 403,
901-906) where it down-regulates at least two target genes, lin-41
(Slack et al., (2000) Molec Cell 5, 659-669) and hbl-1 (Abrahante
et al., (2003) Dev Cell 4, 625-637; Lin et al., (2003) Dev Cell 4,
639-650), mutations which lead to precocious terminal
differentiation of seam cells. To better understand the role of
let-7 in C. elegans seam cell differentiation and its potential
role in humans, additional targets of let-7 were identified. A
computational screen for C. elegans genes was performed with let-7
family complementary sites (LCS) in their 3'UTR (Grosshans, H., et
al., Dev Cell (2005) 8(3):321-30). One of the top scoring genes was
let-60, encoding the C. elegans orthologue of the human oncogene
RAS. 8 LCSs were identified in the 3'UTR of let-60 with features
resembling validated LCSs (Lin et al., (2003) Dev Cell 4, 639-650;
Reinhart et al., (2000) Nature 403, 901-906; Slack et al., (2000)
Molec Cell 5, 659-669; Vella et al., (2004) Genes Dev 18, 132-137)
(FIG. 4A) (SEQ ID Nos. 26, 27, 28, 29, 32, 33, 34 and 35). Many of
the identified sites were found in the 3'UTR of let-60 from the
closely related nematode C. briggsae (Stein et al., (2003) PLoS
Biol 1, E45) (FIG. 4A, FIG. 11 and FIG. 12), suggesting that they
are likely to be biologically significant. An additional three
sites were found in the let-60/RAS coding sequence as well as other
non-conforming 3'UTR sites that may also bind to let-7 family
miRNAs (FIG. 11) (SEQ ID Nos. 60, 61, 62, 63, 64, 65, 66, 67, 68,
69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 and 80).
[0175] let-7(n2853ts) loss of function (10 mutants express reduced
let-7 miRNA and die by bursting at the vulva at the non-permissive
temperature (Reinhart et al., (2000); Slack et al., (2000) Molec
Cell 5, 659-669). Loss of function mutations in two previously
identified targets of let-7, lin-41 and hbl-1, have the property of
partially suppressing the let-7 lethal phenotype (Abrahante et al.,
(2003) Dev Cell 4, 625-637; Lin et al., (2003) Dev Cell 4, 639-650;
Reinhart et al., (2000) Nature 403, 901-906; Slack et al., (2000)
Molec Cell 5, 659-669). It was found that post-embryonic reduction
of function of let-60 by feeding RNA interference (RNAi), also
partially suppressed let-7(n2853) in a reproducible manner. While
5% of let-7 mutants grown on control RNAi survived at the
non-permissive temperature of 25.degree. C. (n=302), 27% of
let-7(n2853); let-60(RNAi) animals survived (n=345). Thus, similar
to other known let-7 targets, let-601f partially suppresses the
let-7(n2853) lethal phenotype, suggesting that let-7 lethality may
at least partially be caused by over-expression of let-60. However
let-60(RNAi) did not appear to suppress the let-7 seam cell
terminal differentiation defect and did not cause precocious seam
cell terminal differentiation. In addition, wild-type animals
subjected to let-60(RNAi) did not display typical lethal and
vulvaless phenotypes associated with let-60 alleles (Beitel et al.,
(1990) Nature 348, 503-509; Han et al., (1990) Genetics 126,
899913; Han and Sternberg, (1990) Cell 63, 921-931). let-60(RNA1)
resulted in approximately 80% knock down of let-60 mRNA (Grosshans,
H., et al., (2005) Dev Cell 8(3):321-30), suggesting that the
remaining let-60 is still sufficient for seam cell differentiation
and vulval development. To verify the specificity of the
let-60(RNAi), it was shown that while let-7(mn112) adults all die,
let-60(n2021); let-7(mn112) adults can live (see Experimental
Procedures). Interestingly, let-7(n2853); let-60(RNAi) animals
delivered a brood and could lay some eggs, suggesting that the
vulval bursting phenotype of let-7 was not suppressed merely
because of the lack of a vulva. let-60 and let-7 are both expressed
in hypodermal seam cells (Dent and Han, 1998 Post-embryonic
expression pattern of C. elegans let-60 ras reporter constructs.
Mech Dev 72, 179-182; Johnson et al., 2003 A microRNA controlling
left/right neuronal asymmetry in Caenorhabditis elegans. Nature
426, 845-849). The let-60 3'UTR was fused behind the Escherichia
coli lacZ gene driven by the hypodermally-expressing col-10
promoter. It was found that reporter gene activity is
down-regulated around the L4 stage (FIG. 5A), around the same time
that let-7 is expressed in the seam cells (Johnson et al., (2003)
Nature 426, 845-849). In contrast, the same reporter gene fused to
an unregulated control 3'UTR was expressed at all stages (FIG. 5A)
(Reinhart et al., (2000) Nature 403, 901-906; Slack et al., (2000)
Molec Cell 5, 659-669; Vella et al., (2004) Genes Dev 18, 132-137;
Wightman et al., (1993) Cell 75, 855-862). It was found that
reporter down-regulation directed by the let-60 3'UTR depended on a
wild-type let-7 gene, since down-regulation failed in let-7(n2853)
mutants (FIG. 5B). Thus, multiple lines of evidence strongly
suggest that let-60 is negatively regulated by let-7. First, the
let-60 3'UTR contains multiple elements complementary to let-7;
second, the let-60 3'UTR directs down-regulation of a reporter gene
in a let-7 dependent manner; third, this down-regulation is
reciprocal to let-7 up-regulation in the hypodermis; and finally,
let-60 loss of function partially suppresses the let-7 lethal
phenotype.
[0176] The let-7 family member mir-84 is dynamically expressed in
the vulval precursor cells. let-60/RAS is best understood for its
role in vulval development (Wang and Sternberg, (2001) Curr Top Dev
Biol 51, 189-220), however let-7 has not been reported to be
expressed in the vulva. In C. elegans, let-7, mir-48, mir-84 and
mir-241 comprise the let-7 family (Lau et al., (2001) Science 294,
858-862; Lim et al., (2003) Genes Dev 17, 991-1008; Reinhart et
al., (2000) Nature 403, 901-906) (FIGS. 10A, 10B). Previous work
demonstrated that a let-7::gfp fusion faithfully recapitulates the
temporal expression of let-7 and is temporally expressed in seam
cell tissues affected in the let-7 mutant (Johnson et al., (2003)
Dev Biol 259, 364-379). The expression pattern of mir-84, the
closest let-7 relative, was examined by fusing 2.2 kilobases (Kb)
of genomic sequence immediately upstream of the miR-84 encoding
sequence to the green fluorescent protein (gfp) gene. mir-84:: gfp
was first observed in the somatic gonad in larval stage 1 (L1). In
L3 animals, strong expression was observed in uterine cells
including the anchor cell (AC), and weak dynamic expression was
observed in the vulval precursor cells (VPCs) (FIGS. 6A-C). VPCs
are multipotent ventral hypodermal cells that generate the vulva
during L3 and later stages (Sulston and Horvitz, (1977) Dev Biol
56, 110-156). VPCs adopt one of three fates depending on EGF
signaling from the AC (Wang and Sternberg, (2001) Curr Top Dev Biol
51, 189-220). The cell closest to the AC, P6.p, receives the most
LIN-3/EGF signal (Katz et al., (1995) Cell 82, 297-307) and adopts
the primary (1.degree.) fate through activation of a RAS/MAPK
signal transduction pathway (Beitel et al., (1990) Nature 348,
503509; Han et al., (1990) Genetics 126, 899-913; Han and
Sternberg, (1990) Cell 10 63, 921-931): P5.p and P7.p receive less
LIN-3 as well as receiving a secondary lateral signal (Sternberg,
(1988) Nature 335, 551-554) from P6.p, and adopt the secondary
(2.degree.) fate: P3.p, P4.p and P8.p adopt the uninduced tertiary
(3.degree.) fate. mir-84: gfp expression was observed during the
early to mid L3 stage in all the VPCs except for P6.p, in which
expression was rarely observed (FIG. 6A). Subsequent VPC expression
in the mid to late L3 stage was restricted to the daughters
(Pn.pxx) of P5.p and P7.p with weaker GFP first appearing in the
P6.p daughters just before their division into P6.pxx. Thereafter,
equivalent expression was observed in the granddaughters (Pn.pxx)
of P5.p, P6.p and P7.p. mir-84:: gfp expression was observed in all
the VPCs except for P6.p at the stage when their fate in vulval
development is determined by signaling from the AC (Ambros, (1999)
Development 126, 1947-1956) suggesting that mir-84 could play a
role in vulval cell fate determination. In the L4 stage, GFP
expression was maintained in the AC and other uterine cells,
appeared weakly in hypodermal seam cells, and was up-regulated to
higher levels in many P5.p-P7.p descendants. A second let-7 family
member, mir-48 was also expressed in non-P6.p VPCs, suggesting the
potential for redundancy between mir-48 and mir-84 in the VPCs.
[0177] mir-84 overexpression causes vulval and seam defects. miR-84
was overexpressed by generating transgenic animals harboring a
multi-copy array of a 3.0 Kb genomic DNA fragment that spans from
2.2 Kb upstream to 0.8 Kb downstream of the miR-84 encoding
sequence (called mir-84(+++)). These animals expressed elevated
levels of miR-84 and displayed abnormal vulval development
phenotypes, including protrusion and bursting of the vulva (40% of
animals, n=40). Consistent with mir-84::gfp expression in seam
cells, it was found that mir-84(+++) animals also exhibited
precocious seam cell terminal differentiation and alae formation in
the L4 stage, a characteristic seen in precocious developmental
timing mutants. In fact, let-7 over-expressing strains also exhibit
precocious seam cell terminal differentiation in the L4 stage
(Reinhart et al., (2000) Nature 403, 901-906). In contrast, animals
carrying an array containing a construct identical to mir-84(+++)
except for a 75 nucleotide (nt) deletion of sequences encoding the
predicted pre-mir-84 (Amir-84 (+++)) did not display any vulval or
seam defects, demonstrating that the phenotypes observed in
mir-84(+++) are dependent on the miR-84 sequence.
[0178] A search was conducted for let-7 family miRNA complementary
sequences (LCS) in the 3'UTRs of all genes known to play a role in
vulval development (Table 2). LCSs have the potential to bind all
members of the let-7 family, including mir-84. Approximately 11
vulval genes contained at least one LCS (Table 2), raising the
possibility that the let-7 family may regulate multiple genes in
the vulva. In this analysis though, let-60/RAS stood out due to the
high number of LCS sites.
TABLE-US-00002 TABLE 2 LCSs in the 3'UTRs of Known Vulva' Genes
Gene Chromosome LCS in 3'UTR eor-1 IV Yes had-1 V Yes let-60 IV Yes
lin-3 IV Yes lin-9 III Yes lin-11 I Yes lin-36 III Yes lin-39 III
Yes lin-45 IV Yes mpk-1 III Yes sem-5 X Yes
[0179] mir-84 overexpression partially suppresses let-60/RAS gain
of function phenotypes. let-60/RAS is active in P6.p following a
lin-3 EGF signal from the anchor cell that activates a MAPK signal
transduction cascade transforming P6.p to the 1.degree. vulval fate
(Han and Sternberg, (1990) Cell 63, 921931). Since mir-84 is
expressed in all VPCs except P6.p, the possibility that mir-84
negatively regulates expression of let-60/RAS in cells not destined
to adopt the 1.degree. fate was examined. Activating mutations in
let-60/RAS cause multiple VPCs (including the non-P6.p VPCs) to
adopt 1.degree. or 2.degree. fates leading to a multivulva (Muv)
phenotype (Han et al., 1990). It was found that over-expression of
mir-84 partially suppressed the Muv phenotype of let-60(gf)
mutations. In the study, 41% (n=51) of let-60(ga89) (Eisenmann and
Kim, (1997) Genetics 146, 553-565) animals displayed a Muv
phenotype, while only 13% (n=168) did so when also over-expressing
mir-84 from a multi-copy array (p<<0.0001 Chi square test).
The same suppression was observed with a second let-60(gf) allele,
let-60(n1046) (Han et al., (1990) Genetics 126, 899-913): 77%
(n=39) of let-60(n) 046) animals displayed a Muv phenotype, while
only 50% (n=113) did so when also over-expressing mir-84
(p<<0.0001 Chi square test). let-60(n) 046) animals displayed
an average of 1.54 pseudovulvae per animal compared to an average
of 0.66 pseudovulvae per let-60(n1046) animal over-expressing
mir-84. For both let-60(gf) alleles, animals exhibiting low
mosaicism for the myo-3:: gfp, co-injection marker, were completely
suppressed, suggesting that the partial suppression was likely due
to mosaicism of the transgeneic array. Neither an empty vector
control (TOPO) (n=111) (p=0.1435 Chi square test), nor the Amir-84
(+++) array (n=129), suppressed the Muv phenotype of let-60(n1046).
For all let-60(gf) experiments, three independent lines behaved
similarly (FIG. 13C).
[0180] The let-60/RAS 3'UTR confines expression to P6.p. The
promoter of let-60/RAS drives reporter expression in all VPCs (Dent
and Han, (1998) Mech Dev 72, 179-182). However, the transgenic
reporters used in this earlier work did not include the let-60
3'UTR. GFP was fused to the let-60 3'UTR and drove GFP expression
in all the VPCs using the VPC-specific lin-31 (Tan et al., (1998)
Cell 93, 569-580) promoter (gfp60). In the late L2 and early L3
stages, GFP was expressed in all the Pn.p cells, but by mid to late
L3 stages, GFP was largely restricted to the P6.p cell (FIG. 6B),
with some expression in the P5.p and P7.p cell descendants. A
similar fusion construct in which the let-60 3'UTR was replaced by
the unregulated unc-54 3'UTR showed GFP expression in all Pn.p
cells (FIG. 6C). Since the lin-31 promoter is active in all Pn.p
cells (Tan et al., (1998) Cell 93, 569-580), this result
demonstrates that the let-60/RAS 3'UTR is sufficient to
down-regulate a reporter gene in the non-P6.p cells.
[0181] The let-60 3'UTR was replaced with the unregulated unc-54
3'UTR in a let-60 genomic DNA fragment. While one could generate
viable lines using a let-60::let-60(+)::let-60 3'UTR construct at
10 ng/.mu.l, it was not possible to generate viable transformants
using this let-60::let-60(+)::unc-54 3'UTR construct, even at 0.1
ng/.mu.l. The results suggest that the removal of the let-60 3'UTR
may severely over-express let-60 and cause lethality.
[0182] let-60/RAS is a likely target of mir-84 in the vulva.
Previous work has demonstrated that VPCs are sensitive to the
levels of let-60/RAS (Beitel et al., (1990) Nature 348, 503-509;
Han et al. (1990)). Animals carrying extra copies of the wild-type
let-60/RAS gene display a Muv phenotype, where non-P6.p VPCs can
adopt the 1.degree. fate. The data strongly suggest that mir-84
negatively regulates let-60 in non-P6.p VPCs. First, mir-84 is
complementary to multiple sites in the let-60 3'UTR. Second, mir-84
is expressed in a reciprocal manner to let-60 in the VPCs. miR-84
is largely absent from P6.p, at the same time as the let-60 3'UTR
confines GFP expression mainly to the P6.p cell lineage. Finally,
mir-84 over-expression partially suppresses the effects of
activating mutations in the let-60 gene. mir-84 modulates the
expression of let-60/RAS in non-P6.p VPCs to reduce flux through
the RAS/MAPK signaling pathway and hence decrease the likelihood
that these cells will also adopt the 1.degree. fate. However,
mir-84 is clearly not the only regulator of let-60/RAS in non-P6.p
cells: daf12(rh61) mutants do not express mir-84 in any VPC (n=60
animals), and yet daf-12(rh61) animals do not display a Muv
phenotype. Other known factors, e.g. synmuv genes, (Berset et al.,
(2001) Science 291, 1055-1058; Ceol and Horvitz, (004) Dev Cell 6,
563-576; Hopper et al., (2000) Mol Cell 6, 65-75; Lee et al.,
(1994) Genes Dev 8, 60-73; Wang and Sternberg, (2001) Curr Top Dev
Biol 51, 189-220; Yoo et al., (2004) Science 303, 663-666; Yoon et
al., (1995) Science 269, 1102-1105) or unknown factors may also
regulate let-60/RAS signaling in these cells.
[0183] The combined results provide strong evidence that let-7 and
mir-84 regulate let-60/RAS expression through its 3'UTR in seam and
vulval cells, cells in which they are all naturally expressed.
Given that the 3'UTR of let-60/RAS contains multiple let-7lmir-84
complementary sites, it is expected that this regulation is
direct.
[0184] let-7 complementary sites in human RAS 3'UTRs. Numerous
miRNAs are altered in human cancers (Cahn et al., (2002) Proc Natl
Acad Sci USA 99, 15524-15529; Cahn et al., (2004) Proc Natl Acad
Sci USA 101, 29993004; Michael et al., (2003) Mol Cancer Res 1,
882-891; Tam et al., (2002) J Virol 76, 4275-4286) and three of the
best understood miRNAs, lin-4 (Lee et al., (1993) Cell 75,
843-854), let-7 (Reinhart et al., (2000) Nature 403, 901-906) and
bantam (Brennecke et al., (2003) Cell 113, 25-36), all regulate
cell proliferation and differentiation. The closest human
homologues of let-7 and mir-84 are the Hs. let-7 family miRNAs
(Lagos-Quintana et al., (2002) Curr Biol 12, 735-739; Pasquinelli
et al., (2000) Nature 408, 86-89). let-60/RAS (SEQ ID No. 87) is
the C. elegans orthologue of human HRAS (SEQ ID No. 84), KRAS (SEQ
ID No. 86), and NRAS (SEQ ID No. 85) (FIGS. 13A-B), which are
commonly mutated in human cancer (Malumbres and Barbacid, (2003)
Nat Rev Cancer 3, 459-465), including lung cancer. It was found
that all three human RAS 3'UTRs contain multiple putative let-7
complementary sites with features of validated C. elegans LCSs (SEQ
ID Nos. 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 55) (FIGS. 4B-D).
Many of these are conserved in rodents, amphibians and fish (FIG.
14 and FIG. 15 (SEQ ID Nos. 39, 97, 98, 99, 100, 101, 102, 103)),
suggesting functional relevance. The presence of putative LCSs in
human RAS 3'UTRs indicates that mammalian let-7 family members may
regulate human RAS in a manner similar to the way let-7 and mir-84
regulate let-60/RAS in C. elegans.
[0185] Human RAS expression is regulated by let-7 in cell culture.
Microarray analysis performed by Ambion, Inc. on six different cell
lines revealed that HepG2 cells express let-7 at levels too low to
detect by microarray analysis. Therefore, by request, Ambion, Inc.,
transfected HepG2 cells with a double-stranded (ds) RNA that mimics
the let-7a precursor. Consistent with the prediction that RAS
expression is negatively regulated by let-7, immunofluorescence
with a RAS-specific antibody revealed that the protein is reduced
by approximately 70% in HepG2 cells transfected with exogenous
let-7a miRNA relative to the same cells transfected with a negative
control miRNA (FIG. 7A). The protein expression levels of GAPDH and
p21.sup.CIP1 were largely unaffected by the transfected let-7a and
negative control pre-miRNAs (FIG. 16A), indicating that let-7a
regulation is specific to RAS. To confirm that the RAS antibody is
specific to RAS protein in the transfected cells, HepG2 cells were
also independently transfected with two exactly complementary
siRNAs targeting independent regions of NRAS. Both siRNAs reduced
cell fluorescence by more than 60% as compared to negative control
siRNA-transfected cells (FIG. 16B).
[0186] It was predicted that cells expressing native let-7 may
express less RAS protein and that inhibition of let-7 may lead to
derepression of RAS expression. To test this, by request, Ambion,
Inc. transfected HeLa cells, which express endogenous let-7
(Lagos-Quintana et al., (2001) Science 294, 853-858; Lim et al.,
(2003) Genes Dev 17, 991-1008), with anti-sense molecules designed
to inhibit the activity of let-7 (Hutvagner et al., (2004) PLoS
Biol 2, E98; Meister et al., (2004) RNA 10, 544-550). Reducing the
activity of let-7 in HeLa cells resulted in an about 70% increase
in RAS protein levels (FIG. 7B). These results, combined with the
reciprocal experiment using pre-let-7 miRNAs discussed above,
indicates that let-7 negatively regulates the expression of RAS in
human cells.
[0187] The 3'UTR of human NRAS and KRAS was fused to a luciferase
reporter gene and these constructs transfected along with
transfection controls into HeLa cells. NRAS contains two naturally
occurring 3'UTRs that utilize alternative polyadenylylation and
cleavage sites, such that one of the 3'UTRs is 2.5 kb longer than
the other. It was found that while the long NRAS 3'UTR strongly
repressed reporter expression compared to an unregulated control
3'UTR (FIGS. 8A-B), the short NRAS 3'UTR led to only slight, but
reproducible, repression of the reporter. The short 3'UTR contains
4 LCSs, while the long form contains 9 LCSs. The KRAS 3'UTR also
repressed the luciferase reporter (FIG. 8A-B), while HRAS was not
tested. The results demonstrate that the 3'UTRs of NRAS and KRAS
contain regulatory information, sufficient to down-regulate the
reporter.
[0188] As with the endogenous RAS experiments described above, the
reciprocal experiment was performed wherein HeLa cells were
transfected with the RAS 3'UTR reporter constructs and the let-7a
anti-sense inhibitor molecule (or a control scrambled molecule).
Cells transfected with the let-7a inhibitor relieved repression
exerted on the reporter relative to the control transfections (FIG.
8C). Since, a loss in the extent of down-regulation is observed
when let-7 is inhibited, these results strongly indicate that let-7
regulates NRAS and KRAS in human cells through their 3'UTRs.
[0189] let-7, RAS and lung cancer. Like let-60/ras, human RAS is
dose-sensitive, since over-expression of RAS results in oncogenic
transformation of human cells (McKay et al., (1986) Embo J 5,
2617-2621; Pulciani et al., (1985) Mol Cell Biol 5, 2836-2841). It
is plausible that loss of miRNA control of RAS could also lead to
over-expression of RAS and contribute to human cancer. Recent work
has mapped let-7 family members to human chromosomal sites
implicated in a variety of cancers (Cahn et al., (2004) Proc Natl
Acad Sci USA 101, 2999-3004). In particular let-7a-2, let-7c and
let-7g have been linked to small chromosomal intervals that are
deleted in lung cancers (Cain et al., (2004) Proc Natl Acad Sci USA
101, 2999-3004), a cancer type in which RAS mis-regulation is known
to be a key oncogenic event (Ahrendt et al., (2001) Cancer 92,
1525-1530; Johnson et al., (2001) Nature 410, 1111-1116).
[0190] miRNA microarray analysis was performed by Ambion, Inc. to
examine expression levels of members of the let-7 gene family in
tissue from twenty-one different cancer patients, including twelve
lung cancer patients with squamous cell carcinomas (stage IB or
IIA). let-7 is poorly expressed in lung tumors, as shown by
expression of let-7 in 21 breast, colon, and lung tumors relative
to associated normal adjacent tissue ("NAT"). Fluorescently labeled
miRNA was hybridized to microarrays that included probes specific
to let-7a and let-7c. Fluorescence intensities for the tumor and
NAT were normalized by total fluorescence signal for all elements
and the relative average signal from the let-7 probes in the tumor
and normal adjacent samples are expressed as log ratios. let-7a and
let-7c had similar profiles suggesting cross-hybridization between
the two closely related miRNAs. let-7 miRNAs were reduced in
expression in a number of the tumors relative to the normal
adjacent tissue samples from the same patients. let-7 was expressed
at lower levels in all of the lung tumor tissues (FIG. 9A), but
only sporadically in other tumor types. A similar finding was
independently discovered (Takamizawa et al., (2004) Cancer Res 64,
3753-3756). On average, let-7 was expressed in lung tumors at less
than 50% of expression in the associated normal lung samples.
Northern analysis was used to measure let-7c in the tumor and NAT
samples for the two patients from which RNA was purified (samples
represented by the first and fifth lung cancer bars in FIG. 9B).
Consistent with the microarray results by Ambion, Inc., northern
analysis verified that the expression of let-7c was 65% lower in
the tumor of patient #1 and 25% lower in the tumor of patient #5.
Seven of eight examined samples also had on average 30% less let-7g
expression in the tumor tissue (FIG. 17). The miRNA arrays were
used to compare the lung tumors and NAT included probes for 167
total miRNAs. The expression of the vast majority of these were
unchanged in the lung tumors indicating that let-7 might be
important in lung cancer. In theory, down regulation of let-7 could
result in up-regulation of RAS and thus induce or accentuate
oncogenesis.
[0191] To test this hypothesis, Ambion, Inc. isolated total RNA and
total protein from the tumor and normal adjacent tissues of three
new lung cancer patients with squamous cell carcinoma. The RNA
samples were split and half was used for northern analysis to
measure let-7c and U6 snRNA. The other halves of the RNA samples
were used for real-time PCR to measure the NRAS mRNA, 18S rRNA, and
B-actin mRNA. The protein samples were used for western analysis to
assess RAS and GAPDH protein levels. RAS protein was present in the
tumors at levels at least ten-fold higher than in the normal
adjacent samples from the same patients. Consistent with the miRNA
array results of Ambion, Inc. for other lung cancer samples, all
three lung tumor samples had 4- to 8-fold lower levels of let-7
than did the corresponding NAT samples. The first and third lung
cancer samples had similar levels of NRAS mRNA in both the tumor
and NAT while the second sample pair had significantly higher
levels of NRAS mRNA in the tumor sample. RAS protein levels
correlate poorly with NRAS mRNA levels but very well with let-7
levels, suggesting that the expression of the oncogene is
significantly influenced at the level of translation, consistent
with the known mechanism of let-7 in invertebrates.
[0192] The reciprocal expression pattern between let-7 and RAS in
cancer cells closely resembles what was seen with let-7 and RAS in
C. elegans and in the human tissue culture experiments. The
correlation between reduced let-7 expression and increased RAS
protein expression in the lung tumor samples indicates that one or
more members of the let-7 gene family regulates RAS expression in
vivo and that the level of expression of the miRNA is an important
factor in limiting or contributing to oncogenesis.
[0193] These results demonstrate that the let-7 miRNA family
negatively regulates RAS in two different C. elegans tissues and in
two different human cell lines. Strikingly, let-7 is expressed in
normal adult lung tissue (Pasquinelli et al., (2000) Nature 408,
86-89), but is poorly expressed in lung cancer cell lines and lung
cancer tissue (Takamizawa et al., (2004) Cancer Res 64, 3753-3756).
The expression of let-7 inversely correlates with expression of RAS
protein in lung cancer tissues, suggesting a possible causal
relationship. In addition, over-expression of let-7 inhibited
growth of a lung cancer cell line in vitro (Takamizawa et al.,
(2004) Cancer Res 64, 3753-3756), suggesting a causal relationship
between let-7 and cell growth in these cells.
[0194] These results demonstrate that the expression of the RAS
oncogene is regulated by let-7 and that over-expression of let-7
can inhibit tumor cell line growth. The combined observations that
let-7 expression is reduced in lung tumors, that several let-7
genes map to genomic regions that are often deleted in lung cancer
patients, that over-expression of let-7 can inhibit lung tumor cell
line growth, that the expression of the RAS oncogene is regulated
by let-7, and that RAS is significantly over-expressed in lung
tumor samples strongly implicates let-7 as a tumor suppressor in
lung tissue and provides evidence of a mechanism forming the basis
for treatment.
Example 2
Expression Patterns of let-7 and mir-125, the lin-4 Homologue
[0195] It was found that both let-7 and mir-125, the lin-4
homologue, are expressed in a variety of adult tissues, with
prominent expression in the lung and brain. Interestingly, both are
expressed at low levels in the pancreas and testis. Past work has
shown that the C. elegans lin-4 and let-7 miRNAs are temporally
expressed (Reinhart, B., et al., (2000) 403:901-906 and Feinbaum,
R. and V. Ambros, (1999) Dev Biol 210(1):87-95). Similarly, it has
been shown that the mammalian homologues for these miRNAs are also
temporally expressed during mouse development. Northern blots
reveal that let-7 and mir-125 have very similar expression
profiles. Both become expressed at around day E9.5. Interestingly,
this coincides approximately with the time of lung organogenesis,
and when other major organs begin to develop.
[0196] An in situ protocol using an oligonucleotide based on the
mouse sequence of the let-7c miRNA, which has been
digoxigenin-labeled, has been developed. Since it has been
confirmed by northern analysis that let-7c is expressed at E12.5,
frozen sections taken from similarly aged embryos have been
analyzed. Preliminary results show let-7 is expressed in the lung
epithelium by in situ. In addition to other cancers, these results
indicate that let-7 is useful as a therapeutic for lung cancer
therapy because let-7 is a natural compound in lung cells.
Example 3
Effect of Inhibition and Overexpression of let-7 in Lung Cancer
Cells
[0197] Inhibition of let-7 function in A549 lung cancer cells via
transfection (with anti-let-7 molecules) causes increased cell
division of A549 lung cancer cells (FIG. 18A), while let-7
over-expression (with transfected pre-let-7) caused a reduction in
A549 cell number (FIG. 18B). These results are consistent with the
tumor suppressing activity of let-7. Moreover, the let-7
over-expression phenotype resembled that caused by MYC
down-regulation (FIG. 18A), suggesting that the effects of let-7 on
cell proliferation may also be through repression of MYC.
Preliminary evidence indicates that MYC is also a direct target of
let-7 in human cells. Therefore, these results indicate that let-7
may be a potential therapeutic in cancers with aberrant expression
of MYC.
Example 4
let-7 Affects Expression of MYC and BCL-2
[0198] let-7 regulates RAS, MYC and BCL-2 protein levels, all three
of which are major cancer oncogenes. Addition of let-7 to HepG2
cells (that do not make let-7 endogenously, reduces the expression
of all three of these important oncoproteins (FIGS. 20, 7A, and 8).
HeLa cells that make endogenous let-7 express increased levels of
all three oncoproteins when transfected with an anti-let-7
inhibitor (FIGS. 20A, B and 7B). These results demonstrate that
let-7 represses expression of these genes. Multiple mir-125 and
mir-143 complementary sites have been identified in the human KRAS
and BCL2 3'U'I'Rs and it is expected these oncomirs may also
regulate these oncogenes in a manner similar to that seen with
let-7. The results indicate that let-7 is a master regulator of
cancer pathways, regulating proliferation (RAS and MYC) and
survival pathways (BCL2). It is possible that let-7 also regulates
telomerase ('PERT) and angiogenesis (VEGF) pathways (Table 1).
Since cancer is the result of multiple genetic mutations, these
results indicate that introduction of let-7 to cancer patients
could repress the expression of multiple oncogenes, and provide an
effective therapy (effectively a one drug cocktail).
Example 5
Effects of Radiation on Cellular miRNA Expression Levels
Experimental Procedures
[0199] Total RNA was collected from cells before and 2, 8 and 24
hours post exposure to 2.5 Gray (Gy) of radiation. Total RNA was
collected from cells using the mirVana kit from Ambion (per
manufacturer's instructions). A total of 10 .mu.g was used for
microRNA microarray by LC Sciences. To confirm the quality of the
RNA a UV test was performed and the samples were enriched for
miRNAs by using a cut-off filter (um100 from microcon-modified
procedure). The microRNAs were then labeled and hybridized to a
microarray chip with multiple repeat regions and a miRNA probe
region, which detects miRNA transcripts listed in Sanger miRBase
release 8.2. This consists of 440 human miRNA sequences. Multiple
control probes were included in each chip. The control probes were
used for quality controls of chip production, sample labeling and
assay conditions. For the in-depth data analysis of our time-point
experiments, LC Sciences performed multi-array normalization, ANOVA
(Analysis of Variance), and clustering analysis. The ANOVA and
clustering analysis were performed on ratio data of individual
arrays (with the multi-array normalization) instead of the often
used intensity data of individual samples. They found this
necessary in order to reveal the rather small miRNA variations
among the samples of different time points. Since there was only
one sample for each time point, they used repeating probe sets of
the arrays to have constructed "groups" that were needed for ANOVA
analysis.
Results
[0200] To determine whether miRNAs are involved in the cellular
response to cytotoxic therapy, miRNA microarrays were utilized to
compare the relative levels of cellular miRNAs before and after
radiation. A lung cancer cell line, A549, in which let-7 levels are
low (Johnson, et al., Cell, 120:635-47 (2005)) and RAS is activated
(Valenzuela, and Groffen, Nucleic Acids Res., 14:843-852 (1986))
was irradiated. The levels of eighty-one miRNAs significantly
changed postirradiation. Significant changes in expression of most
miRNAs was observed as early as two hours post-irradiation, with
most of these early-affected miRNAs returning to their baseline
expression levels by twenty-four hours. Interestingly, each member
of the let-7 family of miRNAs, barring one (let-7g), decreased
significantly by 2 hours post-irradiation (FIG. 21A). Of the 23
miRNAs with decreased expression post-irradiation, 7 (30%) were
members of the let-7 family, a 17-fold enrichment over their
representation on the array (1.8% [8/440 miRNAs on the array]).
Real-time PCR was used to validate the microarray findings for
several let-7 homologues.
[0201] The same microarray analysis was performed in a normal lung
epithelial cell line, CLR2741. The levels of most miRNAs, including
all members of the let-7 family, were significantly different
between these two cell types before radiation. However, both the
normal and tumor cells exhibited similar patterns of miRNA
expression change in response to radiation. The similarity between
the miRNA response postirradiation in the cancerous and normal
epithelial cell lines suggests that a highly conserved global miRNA
response exists in lung cells post-irradiation (FIGS. 21B and 21C),
and further, that miRNAs are critical components of the cellular
response to cytotoxic insult.
Example 6
Effects of let-7 miRNAs on the Radiation Response and Cell Survival
Experimental Procedures
[0202] A549 cells were transfected with 90 nM of the pre-let-7 or
control pre-miR. Several transfection methods with different
carriers were evaluated to compare toxicity versus efficiency, as
measured by a luciferase reporter construct sensitive to let-7
levels (luc fused to the NRAS 3' UTR), and the method with the
least toxicity and most efficient transfection (XtremeGENE, Roche,
data not shown) was selected for transfections. Twenty-four hours
after transfection, cells were treated with increasing doses of
radiation (2.0, 4.0 or 6.0 Gy) and then plated at different
dilutions and grown without being disturbed. Colonies were counted
after two weeks.
Results
[0203] The results obtained in Example 5 above suggested that
altering miRNA levels could be an efficacious approach to alter the
cellular radiation response. Clonogenic cell survival assays
measure all forms of cell death and are the recognized standard for
radiation sensitivity assays (Rockwell, Lab Anim. Sci., 27:931-51
(1977)). Therefore this assay was employed to test the impact of
altering let-7 levels on the radiation response and cell survival.
let-7 over-expression in A549 cell lines causes defects in
proliferation, but does not cause apoptosis in these cells.
Specifically, the impact of let-7b, let-7a, and let-7g on the
radiation response was evaluated because: let-7b drops the most
significantly post-irradiation (FIGS. 1A-1C); let-7a levels have
been implicated in predicting outcome in certain cancers, most
notably lung cancer and; let-7g levels are up-regulated
post-irradiation and significantly changed only in the tumor cell
line (FIGS. 1A-1C). To over-express each of the let-7 homologues of
interest, A549 cells were transfected with synthetic pre-let-7
molecules or control pre-miRNA containing scrambled sequences
(Ambion). Twenty-four hours after transfection, cells were treated
with increasing doses of radiation and then plated at different
dilutions and grown without perturbation. After two weeks, colonies
were counted. Significant radiosensitization was found in cells
treated with pre-let-7b as compared to control pre-miRNA (FIG.
22A). In parallel experiments, anti-miR5 were delivered to A549
cells to specifically decrease let-7 miRNA activity. As expected
from the effects of let-7b over-expression, anti-let-7b caused
significant radioprotection (FIG. 22C). Consistent with the unique
direction of altered expression levels of let-7g post-irradiation,
a unique role for let-7g in the radiation response was identified.
let-7g over-expression protected A549 cells from radiation (FIG.
2B), while anti-let-7g caused radiosensitization of A549 cells
(FIG. 22D), opposite to the effects of let-7a and let-7b. While not
wishing to be bound by theory, it is possible that over-expression
of let-7a and let-7b causes radiosensitization and over-expression
of let-7g radioprotection in part by overcoming the innate
requirement of the cell to down- or up-regulate these miRNAs as
part of the radiation response. However, the molecular mechanisms
of miRNA function in the radiation response may also be related to
alteration in the levels of their targets, such as RAS. These
results indicate that the let-7 miRNA family is important in the
radiation response and that their manipulation is a powerful method
to alter mammalian cell survival post-irradiation.
Example 7
Effects of let-7 miRNAs on the Radiation Response and Cell Survival
in C. Elegans Experimental Procedures
[0204] Methods for culturing, handling and genetic manipulation of
C. elegans were as described by Brenner unless otherwise indicated
(Brenner, Genetics, 77:71-94 (1974)). The animals referred to here
as wild-type C. elegans correspond to the Bristol strain N2.
Strains used in this study were obtained from the C. elegans
Genetics Center (CGC) unless otherwise noted. let-7 over-expressing
strains were generated as described (Reinhart, et al., Nature,
403:901-6 (2000); Esquela-Kerscher, Dev. Dyn., 234:868-77 (2005)).
For synchronization, gravid hermaphrodites were treated as
previously described (Weidhaas, Proc. Natl. Acad. Sci. U.S.A., 103:
9946-51 (2006)). Isolated embryos were treated with radiation as
previously described (Weidhaas, Proc. Natl. Acad. Sci. U.S.A., 103:
9946-51 (2006)). While the doses in these studies may appear high,
when accounting for DNA size using the target theory they were
comparable to human doses. Animals were anesthetized with 5 mM
Levamisole HCl, placed onto 2% agarose pads and examined using
40.times. Nomarski optics. All data points were normalized to their
0 Gy data point to rule out any vulval defects independent of
irradiation. Dose response curves were generated at the first
S-phase radioresistance peak [determined as previously described
(Weidhaas, Proc. Natl. Acad. Sci. USA, 103: 9946-51 (2006))] by
dividing synchronized C. elegans populations into individual
feeding plates and treating each dose point sequentially, with a
start and an end same-dose control sample. For each dose, a minimum
of 100 animals were treated and scored per experiment, and
experiments were repeated 2-4 times. For RNAi after synchronization
animals were placed on plates with the appropriate bacterial strain
containing the plasmid that over-expresses dsRNA from the gene of
interest and grown until appropriate time for radiation. After
irradiation animals were placed on plates with the same bacterial
strain and grown until phenotypic analysis. For statistical
analysis each of the mutant strains were compared against the wild
type using a stratified two-sample Wilcoxon rank sum test.
Stratified t-tests were performed to analyze significance for all
cases. The p-value was based on a two-tailed evaluation of the
data.
Results
[0205] To confirm the ability of let-7 miRNAs to alter the
radiation response in vivo, a powerful C. elegans-based in vivo
model of radiation-induced reproductive cell death ("Radelegans")
was employed (Weidhaas, Proc. Natl. Acad. Sci. U.S.A., 103: 9946-51
(2006)). The tissue model studied is the developing C. elegans
vulva, in which multipotential vulval precursor cells (VPCs)
undergo 3 rounds of cell division and differentiate into the mature
vulva following RAS signaling (Han and Sternberg, Cell, 63:921-931
(1990)). VPCs represent tissue clonogens, considered the critical
and determinant targets of radiation in tumors (Hewitt and Wilson,
British Jour. Cancer, 14:186-94 (1960); Baker and Sanger, Int. J.
Cell Cloning, 9:155-65 (1991)) and die via reproductive cell death
post-irradiation (Weidhaas, Proc. Natl. Acad. Sci. U.S.A., 103:
9946-51 (2006)). Radiation resistance in VPCs depends on RAS
signaling and a normal DNA damage response pathway (Weidhaas, et
al., Cancer Res., 66: 10434-8 (2006)). VPCs show specific
expression of three let-7 paralogues, let-7, mir-48 and mir-84,
that repress RAS expression in this tissue (Esquela-Kerscher, Dev.
Dyn., 234:868-77 (2005)). Upon irradiation, VPCs in strains that
over-express either let-7 or mir-84 were significantly
radiosensitive compared to wild-type animals (FIG. 23A). This is
consistent with results of the analysis of let-7a and let-7b in
lung cancer cells (FIG. 22). Of note, no C. elegans toxicity was
observed in these studies, suggesting that radiosensitivity
mediated by let-7 over-expression is limited to the few actively
dividing tissues, such as VPCs. let-7 mutants could not be analyzed
for radiosensitivity due to gross defects in vulval development.
Instead, animals harboring a mir-84 deletion were analyzed because
they develop without obvious vulval abnormalities. In dose response
experiments, mir-84(tm1304) animals exhibited significant
radioresistance across all radiation doses (FIG. 23B) compared to a
wild-type strain, consistent with the results of the in vitro
analysis of let-7b (FIG. 22). mir-84 has been shown to regulate the
let-60/RAS oncogene that is critical for protection from
reproductive cell death in VPCs. Therefore, the hypothesis was
tested that the radioresistance in the mir-84 mutant was partly due
to let-60/RAS expression. Indeed, let-60/RAS(RNAi) suppressed the
radioresistance in mir84(tm1304) animals (FIG. 23B). Since the
radioresistance of mir-84 mutants depended on a wild-type copy of
let-60/RAS, this strongly suggests that RAS is a critical target of
let-7 in the radioresponse. These studies in C. elegans confirm a
conserved role for miRNAs in the cellular response to cytotoxic
stress, such as radiation.
[0206] It is understood that the disclosed invention is not limited
to the particular methodology, protocols, and reagents described as
these may vary. It is also to be understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present
invention which will be limited only by the appended claims. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
embodiments of the invention described herein. Such equivalents are
intended to be encompassed by the following claims.
Sequence CWU 1
1
110165RNACeanorhabditis elegans 1ugagguagua gguuguauag uuuggaauau
uaccaccggu gaacuaugca auuuucuacc 60uuacc 65261RNADrosophila
melanogaster 2ugagguagua gguuguauag uaguaauuac acaucauacu
auacaaugug cuagcuuucu 60u 61370RNAHomo sapiens 3ugagguagua
gguuguauag uuuggggcuc ugcccugcua ugggauaacu auacaaucua 60cugucuuucc
70422RNACeanorhabditis elegans 4uuauacaacc guucuacacu ca
22520RNACeanorhabditis elegans 5uuauacaacc auucugccuc
20621RNACeanorhabditis elegans 6ugagguagua gguuguauag u
21722DNAArtificialNucleotides common to Let-7 sequences from
various species 7tgaggtagta ggttgtatag tt 22819DNAMus musculus
8tgaggtagta gtttgtgct 19922DNAMus musculus 9tgaggtagta gtgtgtacag
tt 221022DNAMus musculus 10tgaggtagta gtttgtacag ta 221121DNAMus
musculus 11agaggtagta gtttgcatag t 211221DNAMus musculus
12agaggtagta ggttgcatag t 211322DNAHomo sapiens 13tgaggtagta
gattgtatag tt 221422DNAHomo sapiens 14tgaggtagta ggttgtatag tt
221521DNAMus musculus 15tgaggtagga ggttgtattg t 211622DNAHomo
sapiens 16tgaggtagta gattgtatgg tt 221722DNAMus musculus
17tgaggtagta ggttgtgtgg tt 221822DNAHomo sapiens 18tgaggtagta
agttgtattg tt 221922DNACeanorhabditis elegans 19tgaggtagta
tgtaatattg ta 222021DNACeanorhabditis elegans 20tgaggtaggt
gcgagaaatg a 212123DNACeanorhabditis elegans 21tgaggtaggc
tcagtagatg cga 232221DNACeanorhabditis elegans 22tccctgagac
ctcaagtgtg a 212322DNAMus muculus 23tccctgagac cctaacttgt ga
222424DNACeanorhabditis elegans 24tccctgagaa ttctcgaaca gctt
242522DNAMus musculus 25tccctgagac ctttaacctg tg
222626RNACeanorhabditis elegans 26acuugugauc gauccucuuc cgccuc
262725RNACeanorhabditis elegans 27ugcaucgauu gaacuuguuc ucucg
252820RNACeanorhabditis elegans 28uccuucauuc uaauuccuca
202924RNACeanorhabditis elegans 29acaugcaucc gaacccccuc cucg
243022RNACeanorhabditis elegans 30auguuauaau guaugaugga gu
223121RNACeanorhabditis elegans 31uguuauaaug uaugauggag u
213220RNACeanorhabditis elegans 32ugcuuuauuc cccuuccucg
203322RNACeanorhabditis elegans 33uucauacaaa uuauuggccu ca
223423RNACeanorhabditis elegans 34aucugaaagu uuuugcuccc ucg
233519RNACeanorhabditis elegans 35ucauuuuucu cuauuccuc
193625RNAHomo sapiens 36aguucucaga auaacuaccu ccuca 253726RNAHomo
sapiens 37ggcugucuga ccagagaaug caccuc 263818RNAHomo sapiens
38acagcacaaa cacaccuc 183922RNAHomo sapiens 39uugauauguu ggaugaugga
gu 224028RNAHomo sapiens 40agcugugauc agugauuuuc aaaccuca
284132RNAHomo sapiens 41aauugccuuc aauccccuuc ucaccccacc uc
324224RNAHomo sapiens 42aucuaaauac uuacugaggu ccuc 244325RNAHomo
sapiens 43aauuuuccug aggcuuauca ccuca 254430RNAHomo sapiens
44gauugcugaa aagaauucua guuuaccuca 304519RNAHomo sapiens
45aacaggaacu auuggccuc 194625RNAHomo sapiens 46gacaguggaa
guuuuuuuuu ccucg 254719RNAHomo sapiens 47auuaguguca ucuugccuc
194825RNAHomo sapiens 48aaugcccuac aucuuauuuu ccuca 254924RNAHomo
sapiens 49gguucaagcg auucucgugc cucg 245024RNAHomo sapiens
50ggcugguccg aacuccugac cuca 245121RNAHomo sapiens 51gauucaccca
ccuuggccuc a 215228RNAHomo sapiens 52ggguguuaag acuugacaca guaccucg
285328RNAHomo sapiens 53agugcuuaug aggggauauu uaggccuc
285430RNAHomo sapiens 54gaccgugggc cgaggugacu gcagacccuc
305525RNAHomo sapiens 55ggaaccccag cccuuagcuc cccuc 255626RNAHomo
sapiens 56agcccuuagc uccccuccca ggccuc 265723DNAArtificialCommon
nucleotides in Let-7 family miRNAs in C. elegans 57tgaggtaggt
acgttatatg gta 235822DNACeanorhabditis elegans 58tgaggtagta
ggttgtatag tt 225921DNACeanorhabditis elegans 59tgaggtaggt
gcgagaaatg a 216026RNACeanorhabditis elegans 60acuugggauc
gauccucuuc cgccuc 266125RNACeanorhabditis elegans 61ugcaucgauu
gaacuuguuc ucucg 256220RNACeanorhabditis elegans 62uccuucauuc
uaauuccuca 206324RNACeanorhabditis elegans 63acaugcaucc gaacccccuc
cucg 246420RNACeanorhabditis elegans 64ugcuuuaucc cccuuccucg
206522RNACeanorhabditis elegans 65uucauacaaa uuauuggccu ca
226623RNACeanorhabditis elegans 66aucugaaagu uuuugcuccc ucg
236719RNACeanorhabditis elegans 67ucauuuuucu cuauuccuc
196826RNACeanorhabditis elegans 68ugcccaauuu cgccaacuca uuuuca
266930RNACeanorhabditis elegans 69uacauuuuca uuauucauuu aucuguuuua
307021RNACeanorhabditis elegans 70ucgucugcuc gucauuauuu u
217122RNACeanorhabditis elegans 71ucacuuucuc ugacuauuuu ca
227221RNACeanorhabditis elegans 72ucagaauguu uguauugcuu u
217328RNACeanorhabditis elegans 73uacaaauuau uggccucauc uauuuuca
287422RNACeanorhabditis elegans 74ugccggucgu uccguuuauu uu
227518RNACeanorhabditis elegans 75ucguauugca uucauuuu
187621RNACeanorhabditis elegans 76uauaauauuc cuauucuuuu g
217719RNACeanorhabditis elegans 77ugcaaugaua uaaauuuua
197821RNACeanorhabditis elegans 78acggugagac augccuccuc g
217921RNACeanorhabditis elegans 79uaaaugugau uugucaucuc g
218024RNACeanorhabditis elegans 80uaugggaguu gaugaagcau uuua
24811372DNAC. elegans 81atgacggagt acaagcttgt ggtagttgga gatggaggag
ttggtaaatc agcactcacc 60attcaactca tccagaatca ctttgtcgaa gaatacgacc
cgaccataga ggacagctac 120agaaagcagg ttgtgataga cggtgagaca
tgcctcctcg acatattgga taccgccgga 180caagaagaat attcggcgat
gcgtgatcag tacatgagga caggcgaagg atttctgttg 240gttttcgccg
tcaacgaggc taaatctttc gagaatgtcg ctaactaccg cgagcagatt
300cggagggtaa aggattcaga tgatgttcct atggtcttgg tagggaataa
atgtgatttg 360tcatctcgat cagtcgactt ccgaacagtc agtgagacag
caaagggtta cggtattccg 420aatgtcgaca catctgccaa aacgcgtatg
ggagttgatg aagcatttta cacacttgtt 480agagaaattc gcaagcatcg
tgagcgtcac gacaataata agccacaaaa gaagaagaag 540tgtcaaataa
tgtgattcag cgtcgggaat tgcccaattt cgccaactca ttttcagtcg
600tgtcaactcc cacccaatta tcctttctcg tacttttttg gtacattttc
attattcatt 660tatctgtttt atctgaaact tgtgatcgat cctcttccgc
ctctacatac tcttcgaatt 720tccacctttt tttctctatg catcgattga
acttgttctc tcgtctgctc gtcattattt 780tttctccttt tttttcttca
tccttcattc taattcctca tctttcgctt agcccaaatc 840tccattcatt
cataggtgtc aaaactagct gtagtgtgtg atccatatct aaaacatgca
900tccgaacccc ctcctcgttc caaaattggc caactctacc aaaaaaaaca
tcgcaccatt 960tttttttcac tttctctgca tattttcaga atgtttgtat
tgcttttttg atgctttatt 1020ccccttcctc gttttcatac aaattattgg
cctcatctat tttcagaagt tctctgaaaa 1080ttaaattctt ttgcatctgc
cggtcgttcc gtttattttt tctctgtttc ctctcatttt 1140tgtcaagtaa
ttatttctct ttcattaact ataatataga tacaattaga ccccatttct
1200catacatttt ctgaacatct gaaagttttt gctccctcgt attgcattca
tttttctcta 1260ttcctctaca ttttatagtc ctatctgaat ataatattcc
tattcttttg atcaagtttt 1320tattattatt ttattttcaa ggaagtattg
caatgatata aattttaaaa ag 1372821338DNACeanorhabditis briggssae
82atgacggagt acaagcttgt ggtggttgga gatggaggag tgggaaagtc tgctctcact
60atccaactca ttcaaaacca cttcgtcgag gaatacgacc caactataga ggacagctat
120cgaaagcagg tagtgatcga cggagagacc tgcctcctcg atatattgga
tactgctggt 180caagaggagt actcggcgat gcgcgatcag tatatgcgaa
ctggagaggg attccttctg 240gtcttcgccg tcaacgaggc caaatcgttc
gaaaacgtag ccaactacag agagcaaatc 300aggagggtga aggattcaga
tgatgttcca atggttctgg ttggaaacaa gtgcgatttg 360gcttctcggt
cagtggactt ccgaacagtc agcgaaacag ccaagggata cggaatgcca
420aatgtggata cttcagccaa aactcgcatg ggtgtcgatg aggcattcta
cacactcgtt 480cgagagatac gcaagcatcg cgagcgtcac gacaacaaca
aaccacaaaa gaaaaagaag 540tgtcaaatta tgtgattcag ccaaaccctt
tcgccaacga tgtttcgttc atgtcaactc 600gcccagctat cctttctcct
gtgcttcggt acactctttt atctgtttta tctggaattt 660gtgattgatt
ctctcccgac ctatatactc ccatacactt ttatttttct atgcatcgat
720tgaactcgtt cactcgtctg ccacttcaac ccgatattat taaattccgc
acccattttt 780cttccttcta attccgtctt tttcgcttat actatcgttc
ataggtgtaa aaatagtagt 840agtgtgattc atatctgaaa atacatctgg
aacttttccg aaccaaatcc aaaatcacca 900aaaaaaccac accatttttc
actttctttg cttatttccc ctcatattat ttgtattgct 960tccctgatgc
tttattccct cttccgtgcc gtttttggtt tctacccatt tatgctaatc
1020tctgggaacc aaaatctgtt gcatctgccg gtggttcaat tctttttttt
tctccgttac 1080tattgttttt atcaaaccca ttctcttatt aacataacaa
tagaatctct tagacccact 1140ccacaagttt ttttctgaaa catcttttcc
ccattttttt cactttgaat gcttttcctt 1200ccgatttata gctctatctg
aatataatat ttcttaattc ttcttcacat tttttttgta 1260ttttttgcac
caatgatatg aaactgaatt tttgaatatt gatatggaaa cctaaaaata
1320cgtcgttctc ccgtgttt 1338831012DNAArtificialConsensus Sequence
83atgacggagt acaagcttgt ggtgttggag atggaggagt ggaatcgcct cacatcaact
60catcaaacac ttgtcgagaa tacgacccac atagaggaca gctagaaagc aggtgtgatg
120acgggagact gcctcctcga atattggata cgcggcaaga gatatcggcg
atgcggatca 180gtaatggacg ggaggattct tggtttcgcc gtcaacgagg
caaatcttcg aaagtgcaac 240tacggagcaa tggagggtaa ggattcagat
gatgttccat ggttggtgga aaatggattt 300gctctcgtca gtgacttccg
aacagtcagg aacagcaagg gtacggatcc aatgtgaact 360cgccaaaacc
gatggggtga tgagcattta cacactgttg agaatcgcaa gcatcggagc
420gtcacgacaa aaaaccacaa aagaaaagaa gtgtcaatat gtgattcagc
aactttcgcc 480aactttctct gtcaactccc catatccttt ctcttttggt
acatctttat ctgttttatc 540tgaattgtga tgatctctcc tcctaatact
ctcacttttt ttctatgcat cgattgaact 600gttcctcgtc tgccgattat
tttctttttc ttcttctaat tcctttcgct tatctcttca 660ataggtgtaa
aataggtagt gtgatcatat ctaaaatcat cgacttcgcc aaacaatcac
720caaaaaaaca caccattttt cactttcttg ctattttcaa ttttgtattg
ctttgatgct 780ttattccctc tctcttttgg tcctttgtct ctgaaaaatc
tttgcatctg ccggtgttct 840ttttttttct cgttcttttt tttcaaattc
tcttattaac ataaatagac ttagacccat 900ccattttctg aaaattttcc
ctttcatttc tttccttctt tatagctatc tgaatataat 960attctattct
tttcattttt tttatttttt cacaatgata taattttaaa aa 101284189PRTHomo
sapiens 84Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val
Gly Lys1 5 10 15Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val
Asp Glu Tyr 20 25 30Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val
Val Ile Asp Gly 35 40 45Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala
Gly Gln Glu Glu Tyr 50 55 60Ser Ala Met Arg Asp Gln Tyr Met Arg Thr
Gly Glu Gly Phe Leu Cys65 70 75 80Val Phe Ala Ile Asn Asn Thr Lys
Ser Phe Glu Asp Ile His Gln Tyr 85 90 95Arg Glu Gln Ile Lys Arg Val
Lys Asp Ser Asp Asp Val Pro Met Val 100 105 110Leu Val Gly Asn Lys
Cys Asp Leu Ala Ala Arg Thr Val Glu Ser Arg 115 120 125Gln Ala Gln
Asp Leu Ala Arg Ser Tyr Gly Ile Pro Tyr Ile Glu Thr 130 135 140Ser
Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val145 150
155 160Arg Glu Ile Arg Gln His Lys Leu Arg Lys Leu Asn Pro Pro Asp
Glu 165 170 175Ser Gly Pro Gly Cys Met Ser Cys Lys Cys Val Leu Ser
180 18585189PRTHomo sapiens 85Met Thr Glu Tyr Lys Leu Val Val Val
Gly Ala Gly Gly Val Gly Lys1 5 10 15Ser Ala Leu Thr Ile Gln Leu Ile
Gln Asn His Phe Val Asp Glu Tyr 20 25 30Asp Pro Thr Ile Glu Asp Ser
Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45Glu Thr Cys Leu Leu Asp
Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60Ser Ala Met Arg Asp
Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys65 70 75 80Val Phe Ala
Ile Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr 85 90 95Arg Glu
Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val 100 105
110Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys
115 120 125Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile
Glu Thr 130 135 140Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe
Tyr Thr Leu Val145 150 155 160Arg Glu Ile Arg Gln Tyr Arg Met Lys
Lys Leu Asn Ser Ser Asp Asp 165 170 175Gly Thr Gln Gly Cys Met Gly
Leu Pro Cys Val Val Met 180 18586188PRTHomo sapiens 86Met Thr Glu
Tyr Lys Leu Val Val Val Gly Ala Cys Gly Val Gly Lys1 5 10 15Ser Ala
Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30Asp
Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40
45Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr
50 55 60Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu
Cys65 70 75 80Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile
His His Tyr 85 90 95Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp
Val Pro Met Val 100 105 110Leu Val Gly Asn Lys Cys Asp Leu Pro Ser
Arg Thr Val Asp Thr Lys 115 120 125Gln Ala Gln Asp Leu Ala Arg Ser
Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140Ser Ala Lys Thr Arg Gln
Gly Val Asp Asp Ala Phe Tyr Thr Leu Val145 150 155 160Arg Glu Ile
Arg Lys His Lys Glu Lys Met Ser Lys Asp Gly Lys Lys 165 170 175Lys
Lys Lys Lys Ser Lys Thr Lys Cys Val Ile Met 180
18587184PRTCeanorhabditis elegans 87Met Thr Glu Tyr Lys Leu Val Val
Val Gly Asp Gly Gly Val Gly Lys1 5 10 15Ser Ala Leu Thr Ile Gln Leu
Ile Gln Asn His Phe Val Glu Glu Tyr 20 25 30Asp Pro Thr Ile Glu Asp
Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45Glu Thr Cys Leu Leu
Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60Ser Ala Met Arg
Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Leu65 70 75 80Val Phe
Ala Val Asn Glu Ala Lys Ser Phe Glu Asn Val Ala Asn Tyr 85
90 95Arg Glu Gln Ile Arg Arg Val Lys Asp Ser Asp Asp Val Pro Met
Val 100 105 110Leu Val Gly Asn Lys Cys Asp Leu Ser Ser Arg Ser Val
Asp Phe Arg 115 120 125Thr Val Ser Glu Thr Ala Lys Gly Tyr Gly Ile
Pro Asn Val Asp Thr 130 135 140Ser Ala Lys Thr Arg Met Gly Val Asp
Glu Ala Phe Tyr Thr Leu Val145 150 155 160Arg Glu Ile Arg Lys His
Arg Glu Arg His Asp Asn Asn Lys Pro Gln 165 170 175Lys Lys Lys Lys
Cys Gln Ile Met 18088188PRTCeanorhabditis elegans 88Met Arg Glu Tyr
Lys Ile Val Val Leu Gly Ser Gly Gly Val Gly Lys1 5 10 15Ser Ala Leu
Thr Val Gln Phe Val Gln Gly Ile Phe Val Glu Lys Tyr 20 25 30Asp Pro
Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Glu Val Asp Gly 35 40 45Gln
Gln Cys Met Leu Glu Ile Leu Asp Thr Ala Gly Thr Glu Gln Phe 50 55
60Thr Ala Met Arg Asp Leu Tyr Met Lys Asn Gly Gln Gly Phe Val Leu65
70 75 80Val Tyr Ser Ile Thr Ala Gln Ser Thr Phe Asn Asp Leu Met Asp
Leu 85 90 95Arg Asp Gln Ile Leu Arg Val Lys Asp Thr Asp Glu Val Pro
Met Ile 100 105 110Leu Val Gly Asn Lys Cys Asp Leu Glu Asp Glu Arg
Val Val Gly Lys 115 120 125Asp Gln Gly Gln Asn Leu Ala Arg Gln Phe
Gly Ser Ala Phe Leu Glu 130 135 140Thr Ser Ala Lys Ala Lys Ile Asn
Val Ser Glu Val Phe Tyr Asp Leu145 150 155 160Val Arg Gln Ile Asn
Arg Arg Tyr Pro Glu Ser Gly Arg Arg Gln Gly 165 170 175Gln Ser Asn
Lys Gln Cys Cys Ser Cys Val Ile Met 180 18589181PRTCeanorhabditis
elegans 89Met Arg Glu Phe Lys Val Val Val Leu Gly Ser Gly Gly Val
Gly Lys1 5 10 15Ser Ala Leu Thr Val Gln Phe Val Ser Ser Thr Phe Ile
Glu Lys Tyr 20 25 30Asp Pro Thr Ile Glu Asp Phe Tyr Arg Lys Glu Ile
Glu Val Asp Gly 35 40 45Gln Pro Ser Val Leu Glu Ile Leu Asp Thr Ala
Gly Thr Glu Gln Phe 50 55 60Ser Ser Met Arg Asp Leu Tyr Ile Lys Asn
Gly Gln Gly Phe Val Val65 70 75 80Val Tyr Ser Ile Thr Ser Gln Gln
Thr Phe His Asp Ile Arg Asn Met 85 90 95Lys Glu Gln Ile Val Arg Val
Lys Gly Ser Glu Asn Val Pro Ile Leu 100 105 110Leu Val Gly Asn Lys
Cys Asp Leu Ser His Gln Arg Gln Val Arg Ser 115 120 125Glu Glu Gly
Leu Ala Leu Ala Glu Ser Trp Ser Cys Pro Phe Thr Glu 130 135 140Cys
Ser Ala Lys Asn Asn Gln Asn Val Asn Val Thr Phe Ala Glu Ile145 150
155 160Val Arg Glu Met Asn Tyr Val Gln Asn Lys Ser Arg Gln Ser Lys
Ser 165 170 175Cys Cys Ser Leu Met 18090212PRTCeanorhabditis
elegans 90Met Gly Gly Arg Ser Asn Ser Ala Thr Thr Ala Ala Gln Gln
Asn Ala1 5 10 15Val Leu Arg Ile Val Val Val Gly Gly Gly Gly Val Gly
Lys Ser Ala 20 25 30Leu Thr Ile Gln Phe Ile Gln Arg Tyr Phe Val Gln
Asp Tyr Asp Pro 35 40 45Thr Ile Glu Asp Ser Tyr Thr Lys Gln Cys Phe
Val Asp Glu Asp Leu 50 55 60Cys Lys Leu Glu Ile Leu Asp Thr Ala Gly
Gln Glu Glu Phe Ser Thr65 70 75 80Met Arg Glu Gln Tyr Leu Arg Thr
Gly Ser Gly Phe Leu Ile Val Phe 85 90 95Ala Val Thr Asp Arg Asn Ser
Phe Glu Glu Val Lys Lys Leu His Glu 100 105 110Leu Ile Cys Arg Ile
Lys Asp Arg Asp Asp Phe Pro Ile Ile Leu Val 115 120 125Gly Asn Lys
Ala Asp Leu Glu Asn Glu Arg His Val Ala Arg His Glu 130 135 140Ala
Glu Glu Leu Ala His Arg Leu Ser Ile Pro Tyr Ile Glu Cys Ser145 150
155 160Ala Lys Ile Arg Lys Asn Val Asp Glu Ala Phe Phe Asp Ile Val
Arg 165 170 175Leu Val Arg Lys Tyr Gln His Asp Glu Arg Met Pro Ile
His Pro His 180 185 190Asp Asp Arg Lys Leu Glu Ser Pro Ile Lys Leu
Lys Lys Lys Lys Asn 195 200 205Cys Arg Ile Gln
21091211PRTCeanorhabditis elegans 91Met Ser Asn Gly Gly Lys Arg Pro
Pro Glu Asp Asp Ser Lys Leu Pro1 5 10 15Tyr Tyr Lys Leu Val Val Ile
Gly Asp Gly Gly Val Gly Lys Ser Ser 20 25 30Leu Thr Ile Gln Phe Phe
Gln Lys Gln Phe Val Asp Tyr Tyr Asp Pro 35 40 45Thr Ile Glu Asp Gln
Tyr Ile Gln His Cys Glu Ile Asp Gly Asn Trp 50 55 60Val Ile Met Asp
Val Leu Asp Thr Ala Gly Gln Glu Glu Phe Ser Ala65 70 75 80Met Arg
Glu Gln Tyr Ile Arg Gly Gly Arg Gly Phe Leu Leu Val Phe 85 90 95Ser
Val Thr Glu Arg Lys Ser Phe Glu Glu Ala His Lys Leu Tyr Asn 100 105
110Gln Val Leu Arg Val Lys Asp Arg Ser Glu Tyr Pro Val Leu Leu Val
115 120 125Ala Asn Lys Val Asp Leu Ile Asn Gln Arg Val Val Ser Glu
Gln Glu 130 135 140Gly Arg Glu Leu Ala Ala Gln Leu Lys Leu Met Tyr
Ile Glu Thr Ser145 150 155 160Ala Lys Glu Pro Pro Val Asn Val Asp
Ala Ala Phe His Glu Leu Val 165 170 175Arg Ile Val Arg Ser Phe Pro
Ser Asp Glu Gly Asp His Glu Ala Ser 180 185 190Met Ala Ser Val Pro
Arg Thr Lys Lys Arg Lys Asp Lys Gly Lys Cys 195 200 205Leu Ile Ser
210921169DNAHomo sapiens 92caagatactt ttaaagtttt gtcagaaaag
agccactttc aagctgcact gacaccctgg 60tcctgacttc cctggaggag aagtattcct
gttgctgtct tcagtctcac agagaagctc 120ctgctacttc cccagctctc
agtagtttag tacaataatc tctatttgag aagttctcag 180aataactacc
tcctcacttg gctgtctgac cagagaatgc acctcttgtt actccctgtt
240atttttctgc cctgggttct ccacagcaca aacacacctc tgccacccca
ggtttttcat 300ctgaaaagca tttcatgtct gaaacagaga accaaaccgc
aaacgtgaaa ttctattgaa 360aacagtgtct tgagctctaa agtagcaact
gctggtgatt ttttttttct ttttactgtt 420gaacttagaa ctatgctaat
tttggagaaa tgtcataaat tactgttttg ccaagaatat 480agttattatt
gctgtttggt ttgtttataa tgttatcggc tctattctct aaactggcat
540ctgctctaga ttcataaata caaaaatgaa tactgaattt tgagtctatc
ctagtcttca 600caactttgac gtaattaaat ccaactttca cagtgaagtg
cctttttcct agaagtggtt 660tgtagacttc ctttataata tttcagtgga
atagatgtct caaaaatcct tatgcatgaa 720atgaatgtct gagatacgtc
tgtgacttat ctaccattga aggaaagcta tatctatttg 780agagcagatg
ccattttgta catgtatgaa attggttttc cagaggcctg ttttggggct
840ttcccaggag aaagatgaaa ctgaaagcac atgaataatt tcacttaata
atttttacct 900aatctccact tttttcatag gttactacct atacaatgta
tgtaatttgt ttcccctagc 960ttactgataa acctaatatt caatgaactt
ccatttgtat tcaaatttgt gtcataccag 1020aaagctctac atttgcagat
gttcaaatat tgtaaaactt tggtgcattg ttatttaata 1080gctgtgatca
gtgattttca aacctcaaat atagtatatt aacaaattac attttcactc
1140aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1169932302DNAHomo sapiens
93caaagatact tttaaagttt tgtcagaaaa gagccacttt caagctgcac tgacaccctg
60gtcctgactt ccctggagga gaagtattcc tgttgctgtc ttcagtctca cagagaagct
120cctgctactt ccccagctct cagtagttta gtacaataat ctctatttga
gaagttctca 180gaataactac ctcctcactt ggctgtctga ccagagaatg
cacctcttgt tactccctgt 240tatttttctg ccctgggttc ttccacagca
caaacacacc tctgccaccc caggtttttc 300atctgaaaag cagttcatgt
ctgaaacaga gaaccaaacc gcaaacgtga aattctattg 360aaaacagtgt
cttgagctct aaagtagcaa ctgctggtga tttttttttt ctttttactg
420ttgaacttag aactatgcta atttttggag aaatgtcata aattactgtt
ttgccaagaa 480tatagttatt attgctgttt ggtttgttta taatgttatc
ggctctattc tctaaactgg 540catctgctct agattcataa atacaaaaat
gaatactgaa ttttgagtct atcctagtct 600tcacaacttt gacgtaatta
aatccaactt tcacagtgaa gtgccttttt cctagaagtg 660gtttgtagac
ttcctttata atatttcagt ggaatagatg tctcaaaaat ccttatgcat
720gaaatgaatg tctgagatac gtctgtgact tatctaccat tgaaggaaag
ctatatctat 780ttgagagcag atgccatttt gtacatgtat gaaattggtt
ttccagaggc ctgttttggg 840gctttcccag gagaaagatg aaactgaaag
cacatgaata atttcactta ataattttta 900cctaatctcc acttttttca
taggttacta cctatacaat gtatgtaatt tgttttcccc 960tagcttactg
ataaacctaa tattcaatga acttccattt gtattcaaat ttgtgtcata
1020ccagaaagct ctacatttgc agatgttcaa atattgtaaa actttggtgc
attgttattt 1080aatagctgtg atcagtgatt ttcaaacctc aaatatagta
tattaacaaa ttacattttc 1140actgtatatc atggtatctt aatgatgtat
ataattgcct tcaatcccct tctcacccca 1200ccctctacag cttcccccac
agcaataggg gcttgattat ttcagttgag taaagcatgg 1260tgctaatgga
ccagggtcac agtttcaaaa cttgaacaat ccagttagca tcacagagaa
1320agaaattctt ctgcatttgc tcattgcacc agtaactcca gctagtaatt
ttgctaggta 1380gctgcagtta gccctgcaag gaaagaagag gtcagttagc
acaaaccctt taccatgact 1440ggaaaactca gtatcacgta tttaaacatt
tttttttctt ttagccatgt agaaactcta 1500aattaagcca atattctcat
ttgagaatga ggatgtctca gctgagaaac gttttaaatt 1560ctctttattc
ataatgttct ttgaagggtt taaaacaaga tgttgataaa tctaagctga
1620tgagtttgct caaaacagga agttgaaatt gttgagacag gaatggaaaa
tataattaat 1680tgatacctat gaggatttgg aggcttggca ttttaatttg
cagataatac cctggtaatt 1740ctcatgaaaa atagacttgg ataacttttg
ataaaagact aattccaaaa tggccacttt 1800gttcctgtct ttaatatcta
aatacttact gaggtcctcc atcttctata ttatgaattt 1860tcatttatta
agcaaaatgt catattacct tgaaattcag aagagaagaa acatatactg
1920tgtccagagt ataatgaacc tgcagagttg tgcttcttac tgctaattct
gggagctttc 1980acactacgtc atcatttgta aatggaaatt ctgcttttct
gtttctgctc cttctggagc 2040agtgctactc tgtaattttc ctgaggttat
cacctcagtc atttcttttt taaatgtctg 2100tgactggcag tgattctttt
tcttaaaaat ctattaaatt tgatgtcaaa ttagggagaa 2160agatagttac
tcatcttggg ctcttgtgcc aatagccctt gtatgtatgt acttagagtt
2220ttccaagtat gttctaagca cagaagtttc taaatggggc caaaattcag
acttgagtat 2280gttctttgaa taccttaaga ag 2302942297DNARodent
94taagaccctt taaaagttct gtcatcagaa acgagccact ttcaagcctc actgatgccc
60tggttctgac atccctggag gagacgtgtt tctgctgctc tctgcatctc agagaagctc
120ctgcttcctg cttccccaac ttagttactg agcacagcca tctaacctga
gacctcttca 180gaataactac ctcctcactc ggctgtccga accagagaaa
tgaacctgtt tctccccagt 240agttctctgc cctgggtttc ccctagaaac
aaacacacct gccagctggc tttgtcctcc 300gaaaagcagt ttacattgat
gcacgagaac caaactatag acaagcaatt ctgttgtcaa 360cagtttctta
agctctaagg taacaattgc tggtgatttc cccctttgcc cccaactgtt
420gaacttggcc ttgttagttt tgggggaaat gtaaaaatta atcctcttcc
ccgagaatag 480aattagtgtt gctgattgcc tgatttgcaa tgtgatcagc
tatattctat aagctggcgt 540ctgctctgta ttcataaatg caaacatgag
tactgacgta agtgcatccc tagtcttctc 600agctgcatgc aattaaatcc
aacgttcaca acaaagtgcc ttgtcctaac agtgctctgt 660aggcttccgt
tatagttcgt attgaaatag atgtttcaag aaccattgta taggaaagtg
720actatgagcc atctaccttg gagggaaagg tgaatctacc tgatggcaga
tgcttgtata 780tgtacacata tgtacacaaa gacagtttcc ctgtttgcgg
ttctcccagg agaaagaggg 840aactgaaacg attatgacta atttcattta
attctagcta atcttttttt ttttttttgg 900agggggggag taggttacca
cctataaata tttgtaattt cttctagctt actgataatc 960taatagtcaa
tgagcttcca ttataataaa ttggttcata ccaggaagcc ctccatttat
1020agttagtcag atactgtaaa aattggcatg ttattacttt atacctgtga
ttaatgattc 1080ttcaaacctt aaatatagtt attgcaggca ggttatatct
ttgctgcata gtttcttcat 1140ggaaaaaaaa aaatatatat atatatggag
agagtggccc tcagttccca tctcaccatc 1200cctctctttc agcctagatc
agttcaagca tcctatagga gcttgaataa ttatctcagt 1260tgaacaaacc
atggtgctaa tggaccaggt catggtttca aaacttgaac aagccagtta
1320gatcacagag aaaacagttc atccatattt gcctccctgc ctattactcc
tgcttgtaga 1380cttttgcctg atgcctgctg ttcgagctat aaggataaaa
gttagtgtgg ttctacacca 1440ggactgggaa tgcctggtga gctgttgggt
aagcctagac acctttacat tttcagaccc 1500gtatttttag ccccatggaa
actgaagcca gagttcacac ctccatctct tcccccatta 1560gataaatgtt
cttaatctat atagcttttt aaaagtattt aaaacatgtc tataagttag
1620gctaccaact aacaaaagct gatgtgtttg ttcaaataaa gaggtatcct
ttactacttg 1680agaaaagaat gtaaaatgcc attaattgtt gtcatgtaga
agtttgatat ttgtggtaat 1740gccctgataa ttcattggtg agtttgttag
tcatggtgat acttaaaata taactcatct 1800cagtaatttc aatgaaaaca
taaaatggga tgccttgatt gaaaaaagca aacctaattc 1860caaaatgacc
attttctctt ctgatcttac gacacctaaa aatctgagat ccttgggatt
1920catttgttta taacaggaac ttgctatgta atcttggctg gcctcaaact
cacaatgctc 1980ttcctgcatc agtctcaaaa tgatgggatt acaggcacat
gccaccacac acacctgatc 2040tggtttctaa tgaattttta ttgttaagca
aatccccatc accttgaaac taatcagaag 2100agggaagaaa catatgttgt
gctcctcagt gctaatgctg ggatctttca ccaggggttt 2160gcattcttaa
gtaaactgct gcctttttac aacataggct cagtcatcct cctgaagctg
2220cttgagacca acacttggtc ttgttctttt ttaatgtgtg ttatgactgg
tggtggatct 2280ctaaaaagtt tattaaa 2297952225DNARodent 95aagttctgtc
atcagaaaag agccactttg aagctgcact gatgccctgg ttctgacatc 60cctggaggag
acctgttcct gctgctctct gcatctcaga gaagctcctg cttcctgctt
120ccccgactca gttactgagc acagccatct aacctgagac ctcttcagaa
taactacctc 180ctcactcggc tgtctgacca gagaaatgga cctgtctctc
ccggtcgttc tctgccctgg 240gttcccctag aaacagacac agcctccagc
tggctttgtc ctctgaaaag cagtttacat 300tgatgcagag aaccaaacta
gacatgccat tctgttgaca acagtttctt atactctaag 360gtaacaactg
ctggtgattt tcccctgccc ccaactgttg aacttggcct tgttggtttg
420gggggaaaat gtcataaatt actttcttcc caaaatataa ttagtgttgc
tgattgattt 480gtaatgtgat cagctatatt ccataaactg gcatctgctc
tgtattcata aatgcaaaca 540cgaatactct caactgcatg caattaaatc
caacattcac aacaaagtgc ctttttccta 600aaagtgctct gtaggctcca
ttacagtttg taattggaat agatgtgtca agaaccattg 660tataggaaag
tgactctgag ccatctacct ttgagggaaa ggtgtatgta cctgatggca
720gatgctttgt gtatgcacat gaagatagtt tccctgtctg ggattctccc
aggagaaaga 780tggaactgaa acaattacaa gtaatttcat ttaattctag
ctaatctttt tttttttttt 840ttttttggta gactatcacc tataaatatt
tggaatatct tctagcttac tgataatcta 900ataattaatg agcttccatt
ataatgaatt ggttcatacc aggaagccct ccatttatag 960tatagatact
gtaaaaattg gcatgttgtt actttatagc tgtgattaat gattcctcag
1020accttgctga gatatagtta ttagcagaca ggttatatct ttgctgcata
gtttcttcat 1080ggaatatata tctatctgta tgtggagaga acgtggccct
cagttccctt ctcagcatcc 1140ctcatctctc agcctagaga agttcgagca
tcctagaggg gcttgaacag ttatctcggt 1200taaaccatgg tgctaatgga
ccgggtcatg gtttcaaaac ttgaacaagc cagttagcat 1260cacagagaaa
cagtccatcc atatttgctc cctgcctatt attcctgctt acagactttt
1320gcctgatgcc tgctgttagt gctacaagga taagcttgtg tggttctcac
caggactgga 1380agtacctggt gagctctggg gtaagcctag atatctttac
attttcagac ccttattctt 1440agccacgtgg aaactgaagc cagagtccat
acctccatct ccttcccccc ccaaaaaaat 1500tagattaatg ttctttatat
agctttttta aagtatttaa aacatgtcta taagttaggc 1560tgccaactaa
caaaagctga tgtgtttgtt caaataaaga ggtatccttc gctactcgag
1620agaagaatgt aaaatgccat tgattgttgt cacttggagg cttgatgttt
gccctgataa 1680ttcattagtg ggttttgttt gtcacatgat acctaagatg
taactcagct cagtaattct 1740aatgaaaaca taaattggat accttaattg
aaaaaagcaa acctaattcc aaaatggcca 1800ttttctcttc tgatcttgta
atacctaaaa ttctgaggtc cttgggattc ttttgtttat 1860aacaggatct
tgctgtgtag tcctagctgg cctcaaactc acaatactct tcctggatca
1920atctcccaag tgctgggatt acaggcacat tccaccacac acacctgact
gagctcgttc 1980ctaatgagtt ttcattaagc aaattcccca tcaccttgaa
actaatcaga agggggaaca 2040aacatttgct atgctcctga gtgctaacac
tgggctcatt cacatggggt ttgcattcct 2100aggcaaacta aactgctgcc
ttttacaaca aggctcagtc atcttcctga agctgctgag 2160accagcactt
ggtcttgttt tgttttaata tgtcttatga ctggtggtgg atccgtcgac 2220ctgca
222596464DNARodent 96caagatattt aacaaagttc tatcagaaaa gagccacttt
caagctgcac tgataccctg 60gtcctgactt ccctggagga gaagtatccc tgttgctctc
ttcatctcag agaagctcct 120gctgtttgtc cacctctcag tgtatgagca
cagtctctgc ttgagaactt ctcagaataa 180ctacctcctc acttggttgt
ctgaccagag aaatgcacct cttgttaatt ccccaataat 240tttctgccct
gggctctccc caacaaaaaa caaacacttc tgccatccaa aaagcaactt
300ggtctgaaac agaaccaaac tgtagattga aattctctta aaaagtcttg
agctctaaag 360ttagcaaccg ctggtgattt ttattttcct ttttattttt
gaacttggaa ctgacctatg 420ttagattttg gagaaatgtc ataaagtact
gttgtgccaa gaag 4649725RNAXenopus Laevis 97aacacuuuuc aauaccacuu
accuc 259821RNAXenopus Laevis 98aauucuugcu guuaugccuc a
219920RNAXenopus Laevis 99aacaaauugu ugagcaccuc
2010023RNACeanorhabditis elegans 100uugauauguu ggauggaugg agu
2310121RNADanio rerio 101aauuuaucac auuuacccuc a 2110221RNADanio
Rerio 102aaugaauaua uuauuuccuc a 2110321RNACeanorhabditis elegans
103uugauauguu gaugauggag u 21104407DNARodent 104tcaaatgcat
ggtcaagtgc aacctcacaa ccttggctgg gtcttaggat tgaaaggttt 60agccataatg
taaactgcct caaatggaat tttgggcata aaagaagctt gccatctttt
120tgtttgtttg ttttccttta acagatttgt atttaagaat tgtttttaaa
aaatgtgtca 180agtttacccc gttttcctgt gtaaatatgg ccataacttt
aataaaacgt ttatagcagt 240tatacaagaa
ttcaaaccat gtattataaa ccataatttt ttttatttaa gtacattttc
300tgattttttc tattgttttt agaaaaaata aaatacgtgg caaatatata
attgagccaa 360atcttaagtt gtgagtgttt tgtttttctt gccttttttt tctattt
407105378DNAHomo sapiens 105tcaaatgcat gatcaaatgc aacctcacaa
ccttggctga gtcttgagac tgaaagattt 60agccataatg taaactgcct caaattggac
tttgggcata aaagaagctt accatctttt 120ttttttcttt aacagatttg
tatttaagaa ttgtttttaa aaaaatttta agatttacac 180aatgtttctc
tgtaaatatt gccataactt taataaaacg tttatagcag ttacacagaa
240tttcaatcct agtatatagt acctagtatt ataggtacta taaaccctaa
ttttttttat 300ttaagtacat tttgtgattt ttttctattg tttttagaaa
aaataaaata actggcaaat 360aaaaaaaaaa aaaaaaaa 378106326DNARodent
106aaaatgcatg ctcaaagcct aacctcacaa ccttggctgg ggctttggga
cttcagccat 60aatgttaact gcctcaaagt taaggcataa aagaagcttc ccatcttctt
tctttttcct 120ttaacagatt tgtatttaat tgtttttttt aaaaaaatct
tccggtgtac atagggcctt 180aactttaata aaacgtttat aacagttata
caagatttta agacatgtat gataaaccat 240aatttttttt atttaaagac
cttttctgat ttttttctat tgtttttaga aaaaataaaa 300taattggaaa
aaatataatt gagcca 326107326DNARodent 107ttaaaatgca tgctcaaagc
ctaacctcac aaccttggct ggggctttgg gactgtaagc 60ttcagccata attttaactg
cctcaaactt aaatagtata aaagaagctt cccatctttt 120ttctttttcc
ttttaacaga tttgtattta attgtttttt taaaaaaatc taaaatctat
180ccaattttcc catgtaaata gggccttaac tttaataaaa cgtttataac
agttacaaaa 240gattttaaga catgtaccat aatttttttt atttaaagac
attttctgat ttttttctat 300tgtttttaga aaaaaataaa ataatt
32610824RNAHomo sapiens 108aaugcaugau caaaugcaac cuca
2410922RNAHomo sapiens 109agccauaaug uaaacugccu ca
2211021RNACeanorhabditis elegans 110uuguauguug gaugauggag u 21
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References